U.S. patent application number 14/460841 was filed with the patent office on 2014-11-27 for swine genetically modified with specificity for ldl-r knockout.
This patent application is currently assigned to Recombinetics. The applicant listed for this patent is Recombinetics, Inc.. Invention is credited to Daniel F Carlson, Scott C Fahrenkrug.
Application Number | 20140351965 14/460841 |
Document ID | / |
Family ID | 46719944 |
Filed Date | 2014-11-27 |
United States Patent
Application |
20140351965 |
Kind Code |
A1 |
Fahrenkrug; Scott C ; et
al. |
November 27, 2014 |
SWINE GENETICALLY MODIFIED WITH SPECIFICITY FOR LDL-R KNOCKOUT
Abstract
Compositions and methods for swine LDL-R gene knockouts.
Inventors: |
Fahrenkrug; Scott C;
(Minneapolis, MN) ; Carlson; Daniel F; (Woodbury,
MN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Recombinetics, Inc. |
Saint Paul |
MN |
US |
|
|
Assignee: |
Recombinetics
|
Family ID: |
46719944 |
Appl. No.: |
14/460841 |
Filed: |
August 15, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13404662 |
Feb 24, 2012 |
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14460841 |
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61446651 |
Feb 25, 2011 |
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Current U.S.
Class: |
800/17 ; 435/325;
435/352; 435/455; 435/462 |
Current CPC
Class: |
A01K 2217/072 20130101;
A01K 67/0276 20130101; A01K 2217/075 20130101; A01K 67/0275
20130101; A01K 2227/101 20130101; C12N 15/85 20130101; A01K
2227/108 20130101; C12N 15/907 20130101; C12N 15/8509 20130101;
A61D 19/04 20130101 |
Class at
Publication: |
800/17 ; 435/462;
435/352; 435/325; 435/455 |
International
Class: |
A01K 67/027 20060101
A01K067/027; C12N 15/85 20060101 C12N015/85; C12N 15/90 20060101
C12N015/90 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under grant
nos. 1R41HL108440-01 "Development of Porcine Genetic Models of
Atherosclerosis" awarded by the National Institutes of Health. The
United States government has certain rights in the invention.
Claims
1. A method of making a genetically modified swine comprising
exposing a primary cell in vitro or an embryo to a target-specific
nuclease that specifically binds to an LDL-R gene.
2. The method of claim 1 comprising the primary cell, with the
primary cell being a low-passage cell.
3. The method of claim 2 wherein the primary cell is exposed to the
TALEN in the in vitro culture.
4. The method of claim 3 wherein the TALEN is a left TALEN and
further comprising a right TALEN that cooperates with the left
TALEN to make a double strand cut in a DNA.
5. The method of claim 1 comprising introducing, into the cell or
embryo, a plasmid or an mRNA encoding the target-specific
nuclease.
6. The method of claim 1 wherein the target-specific nuclease
comprises a zinc finger.
7. The method of claim 1 wherein the target-specific nuclease
comprises a fusion protein of TAL effectors and a nuclease
(TALEN).
8. The method of claim 1 further comprising exposing the cell or
the embryo to a vector encoding a reporter, with said vector
encoding the reporter being free of sequences that encode the
targeted nuclease.
9. The method of claim 8 wherein the vector comprises a plasmid or
a transposon.
10. The method of claim 8 further comprising choosing a cell or an
embryo that expresses the reporter for further processing to use to
make a genetically modified swine.
11. The method of claim 1 comprising the cell, wherein the cell is
chosen from the group consisting of a primary cell, a primary
somatic cell, a zygote, a primordial germ cell, a stem cell, and a
zygote.
12. A genetically modified swine comprising a genomic knockout of a
low density lipoprotein receptor (LDL-R) gene.
13. The swine of claim 12 being free of exogenous reporter
genes.
14. The swine of claim 12 being a homozygous knockout for the LDL-R
gene.
15. The swine of claim 12 being a heterozygous knockout for the
LDL-R gene.
16. The swine of claim 12 further comprising an exogenous reporter
gene on a chromosome that does not comprise the LDL-R gene.
17. The swine of claim 12 being free of genetic modifications other
than the knockout of the LDL-R gene.
18. The swine of claim 12 with the fourth exon of the LDL-R gene
being disrupted.
19. A cell or an embryo comprising a nucleic acid fragment encoding
a targeted nuclease and a genetic modification at a DNA site that
is specifically bound by the targeted nuclease, wherein the cell
comprises a primary cell isolated from an animal tissue, and
wherein the cell and the embryo are chosen from the group
consisting of artiodactyls, swine, bovine, fish, rabbit, and
livestock.
20. The cell or embryo of claim 19 wherein the targeted nuclease
comprises a fusion protein of TAL effectors and a nuclease
(TALEN).
21. The cell or embryo of claim 19 wherein the targeted nuclease
comprises a zinc finger nuclease.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation of U.S. Ser. No.
13/404,662 filed Feb. 24, 2012, which claims priority to U.S. Ser.
No. 61/446,651 filed Feb. 25, 2011, which are hereby incorporated
by reference herein for all purposes.
TECHNICAL FIELD
[0003] The technical field relates to creation of genetically
modified animals, for example, livestock animals with functional
traits.
BACKGROUND
[0004] Transcription activator-like (TAL) effector sequences can be
assembled to bind DNA targets with specificity by assembling
sequences of repeat variable-diresidue (RVDs). Fusion proteins of
TAL effectors and nucleases (TALENs) can make targeted
double-stranded breaks in cellular DNA that can be used to make
specific genetic modifications to cells. TALENs have been reported
as useful to generate stably modified human embryonic stem cell and
induced pluripotent stem cell clones, to generate knockout C.
elegans, and knockout zebrafish.
SUMMARY
[0005] Transcription Activator-Like (TAL) effectors (TALEs) fused
with nucleases (TALENs) have been reported for use in genetic
modification of cells. These reports have generally focused on
plant cells, transformed (immortalized) animal cell lines, or mouse
embryonic stem cells. FIG. 1 depicts some of the features of a
TALEN-based genetic engineering system.
[0006] One embodiment set forth herein is a method of creating a
genetic modification comprising exposing a primary cell in an in
vitro culture or an embryo to a nucleic acid encoding a TALEN,
wherein the cell is a livestock cell and the embryo is a livestock
embryo. The method may comprise exposing the primary cell to the
nucleic acid in the in vitro culture. The nucleic acid encoding a
TALEN may comprise an mRNA. The TALEN may be a left TALEN and the
method may further comprise a right TALEN that cooperates with the
left TALEN to make a double strand cut in a DNA. A second vector
may comprise the nucleic acid encoding the TALEN. The method may
further comprise exposing the cell or the embryo to a vector
encoding a reporter. The method may comprise a vector encoding a
selection marker. The reporter may comprise a selection marker. The
reporter may require a transposition event performed by a
transposase to be expressed. The method may comprise choosing a
cell or embryo that expresses the reporter for further processing.
The method may comprise wherein the cell is chosen based on
elimination of cells from the culture that do not express the
reporter. The method may comprise wherein the reporter comprises a
fluorescent biomolecule. The method may comprise wherein the embryo
is chosen based on expression of the reporter. The method may
comprise herein the cell is chosen based on expression of the
reporter. The method may comprise wherein the vector comprises a
plasmid. The method may comprise wherein the vector comprises a
transposon that encodes the reporter, and further comprising a
vector that encodes a transposase that recognizes the transposon.
The method may comprise herein the transposase is chosen from the
group consisting of Sleeping Beauty, Frog Prince, Tol2, Minos,
Hsmar, Plaice, and Passport. The method may comprise choosing a
cell or embryo that expresses the reporter and using the cell or
embryo to create a genetically modified animal, with the genetic
modification being at a DNA site specifically bound by the TALEN.
The method may comprise breeding the animal and selecting progeny
that express the genetic modification and are free of exogenous
reporters. The method may comprise wherein the animals are
homozygous bi-allelic for the modification, or heterozygous for the
modification, or heterozygous bi-allelic for the modification. The
method may comprise cloning a genetically modified animal from the
cell of the culture wherein the cell comprises a genetic
modification at a DNA site specifically bound by the TALEN. The
method may comprise herein the cell is used to clone the animal by
somatic cell nuclear transfer or chromatin transfer. The method may
comprise wherein the genetic modification is chosen from the group
consisting of an insertion, a deletion, the insertion of an
exogenous nucleic acid fragment, an inversion, a gene conversion to
natural allele, gene conversion to a synthetic allele, and a gene
conversion to a novel allele. The method may comprise delivering a
recombinase to the cell or embryo. The method may comprise wherein
the recombinase being is delivered by direct injection or by a
vector that encodes the recombinase as a protein, an mRNA, or by a
vector that encodes the recombinase. The method may comprise
wherein the recombinase combines with a nucleic acid to form a
filament, with the nucleic acid providing a template for homology
dependent repair. The method may comprise herein the recombinase is
chosen from the group consisting of RecA, recA803, uvsX, recA
mutants, recA-like recombinases, Cre recombinase, Hin recombinase,
RAD51, Tre, FLP, RuvC, DST2, KEM1 XRN1, STPa/DST1, and HPP-1. The
method may comprise herein the cell is a zygote, or wherein the
embryo is a blastocyst.
[0007] Another embodiment set forth herein is a cell or an embryo
comprising a nucleic acid fragment encoding a TALEN and a genetic
modification at a DNA site that is specifically bound by the TALEN,
wherein the cell comprises a primary cell isolated from an animal
tissue, and wherein the cell and the embryo are chosen from the
group consisting of artiodactyls, swine, bovine, fish, rabbit, and
livestock. The primary cell may be, e.g., exposed to the nucleic
acid in an in vitro culture. The nucleic acid encoding a TALEN may
comprise an mRNA. The cell or embryo may comprise a vector encoding
a reporter. The cell or embryo may comprise a vector encoding a
selection marker. The cell or embryo may comprise wherein the
reporter comprises a selection marker. The cell or embryo may
comprise a vector encoding a reporter, and a vector that encodes
the TALEN. The cell or embryo may comprise a vector encoding a
transposase, with the reporter and the TALEN being transposons
encoded by their respective vectors. The cell or embryo may be
homozygous bi-allelic for the genetic modification. The cell or
embryo may comprise an exogenous recombinase.
[0008] An embodiment of the invention is a genetically modified
livestock animal comprising a genetic modification chosen from the
group consisting of an insertion, a deletion, the insertion of an
exogenous nucleic acid fragment, an inversion, a gene conversion to
natural allele, a gene conversion to a synthetic allele, and a gene
conversion to a novel allele, with the animal being free of
exogenous reporter genes. The livestock animal may be chosen from
the group consisting of artiodactyls, cows, swine, sheep, goats,
rabbits, poultry, and fish. The genetic modification may comprise a
deletion that comprises deletion of 500 basepairs, or a
megabasepair, or an amount of a chromosome ranging from about 10%
to about 90%; artisans will immediately appreciate that all the
ranges and values within the explicitly stated ranges are
contemplated. The genetic modification may comprise a deletion that
comprises a megabase of chromosomal DNA. The genetic modification
may comprise an inversion that comprises 500 basepairs. The genetic
modification may comprise an inversion, with the inversion encoding
a genetic trait. The livestock animal may be comprising a bovine
line of animals that are not Belgian Blue cattle, wherein the
genetic modification comprises a myostatin mutation deletion
prevalent in Belgian Blue cattle.
[0009] An embodiment is a method of genetically modifying a
livestock animal comprising selecting a trait and inverting a
region of DNA that encodes the trait in a cell or embryo that is
used as a progenitor for a livestock animal. The genetic
modification may comprise an in version or a deletion, with the
inversion or the deletion comprising 500 basepairs, or a
megabasepair, or an amount of a chromosome ranging from about 10%
to about 90%; artisans will immediately appreciate that all the
ranges and values within the explicitly stated ranges are
contemplated.
[0010] An embodiment is a method of genetically modifying an animal
comprising exposing a progenitor cell or embryo to a first TALEN at
a first site and a second TALEN at a second site, with the region
between the site being deleted. The first TALEN may comprise a
first TALEN pair and the second TALEN may comprise a second TALEN
pair. In the case using the progenitor cell, for example, somatic
cell nuclear transfer or chromatin transfer from the progenitor
cell may be used to create the animal. The method may be a method
comprising introducing the TALENs into the progenitor cell and also
introducing a vector comprising a reporter, and selecting the
progenitor cell or embryo for creation of the animal only if the
reporter is expressed.
[0011] An embodiment is a method of transfer of an allelic mutation
from a first livestock breed to a second livestock breed comprising
introducing a TALEN into a cell or embryo of the second livestock
breed in a presence of a nucleic acid that encodes the allelic
mutation of the first livestock breed wherein the allelic mutation
is copied into the cell or the embryo, and creating an animal of
the second breed from the cell or the embryo. The copying process
may comprise an HDR template. The allelic mutation may comprise,
e.g., a myostatin mutation allele present in Belgian Blue cattle.
The TALEN may be introduced as a protein or encoded by an mRNA or
by a vector. The method may be comprising introduction of a
reporter vector independently of the TALEN. The allele may be
linked to a trait of the first livestock breed. The method may be
further comprising delivering a recombinase to the cell or embryo.
The recombinase may be delivered as a protein, an mRNA, or by a
vector that encodes the recombinase. The recombinase may be used to
combine with the nucleic acid to form a filament.
[0012] An embodiment is a genetically modified livestock animal
comprising a first breed of livestock animal comprising an allele
of a second breed of livestock animal, wherein the first breed of
livestock animal is free of meiotic recombination with the second
breed of animal. The animal may be, e.g., chosen from the group
consisting of swine, cows, sheep, goats, chickens, rabbits, and
fish. As an example the first breed is Wagyu cattle and the second
breed is Belgian Blue cattle. The allele may be chosen from the
group consisting of an insertion, a deletion, a polymorphism, and a
single nucleotide polymorphism. The animal may be free of exogenous
marker genes. The animal may be comprising a plurality of the
alleles. The allele or alleles may be linked to a quantitative
trait or qualitative trait of the second breed.
[0013] The following patent applications are hereby incorporated
herein by reference for all purposes; in case of conflict, the
specification is controlling: US 2010/0146655, US 2010/0105140, US
2011/0059160, and US 2011/0197290.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1 is an illustration of a TALEN and genetic
modifications caused by the same.
[0015] FIG. 2 is an illustration of TALENs operating at a plurality
of DNA loci.
[0016] FIG. 3 TALEN activity in bovine embryos. An experimental
overview is given in panel (a). TALENs are designed to opposing
strands of the DNA target such that the FokI nuclease homodimeric
monomers are able to dimerize and cleave DNA between the two
monomers. Bovine in vitro-produced zygotes are injected with TALEN
mRNA on day 1 (D1) and cultured in vitro to blastocyst formation.
Individual blastocysts (blasts) are collected on day 8, subjected
to whole genome amplification (WGA) and analyzed for indels by PCR
amplification and Cel-I (SURVEYOR Nuclease, Transgenomics)
treatment. Panel b) SURVEYOR Nuclease treatment for analysis of
indels in bovine embryos mediated by ACAN12 TALENs. The amplicon
length and predicted SURVEYOR cleavage products that are indicative
of indels, is shown above. Panel c) SURVEYOR Nuclease treatment for
analysis of indels in porcine zygotes mediated by p65 TALENs.
[0017] FIG. 4A. Deletions and insertions sequenced from bovine
embryos treated with ACAN12 TALENs. The wild-type sequence is shown
with TALEN binding sites underlined. Both deletion and insertion
events were identified.
[0018] FIG. 4B. Deletions and insertions sequenced from porcine
embryos treated with ACAN12 TALENs. The wild-type sequence is shown
with TALEN binding sites underlined. Both deletion and insertion
events were identified.
[0019] FIG. 5 Comparison of TALEN scaffold for gene editing in
livestock fibroblasts. Panel a) A diagram of TALEN scaffolds tested
in this experiment. Each scaffold (+231, Christian et. al. 2010
{Christian, 2010} and +63, Miller et. al. 2011 {Miller, 2011})
contains a SV40 nuclear localization signal (NLS) and has a
C-terminal fusion of the FokI homodimer domain. Numbering is
relative to the DNA binding domain. The amino acid prior to the
first repeat variable diresidue repeat (RVD) is labeled "-1" and
the amino acid following the last RVD repeat is labeled "+1". Panel
b) The SURVEYOR assay was conducted on fibroblasts transfected with
either DMDE7.1 or ACAN12 TALEN pairs. Scaffold and temperature
treatment is indicated above the gel and percent NHEJ is indicated
below. Abbreviations, NT=not treated. Panel c) Activity of four
additional TALEN pairs with either the +231 or +63 scaffold.
[0020] FIG. 6 Deletions and insertions sequenced from cells treated
with ACAN12 TALENs. The wild-type ACAN12 sequence is displayed in
italics and the left and right (complimentary) TALEN-recognition
sequences are underlined. Inserted nucleotides are highlighted in
grey and mismatch nucleotides are denoted by lower-case text.
[0021] FIG. 7 Transposon co-selection for indel enrichment. An
experimental timeline is shown in panel (a). Day zero (D0), cells
are transfected with a mixture of plasmids including an expression
cassette for each TALEN, a transposon encoding a selection marker,
and a transposase-expression cassette. The TALEN plasmid is the
major component (4-fold excess by mass) of each transfection.
Transfected cells are cultured for 3 days at either 30 or
37.degree. C. prior to splitting, collection of a sample for
SURVEYOR assay and re-plating for extended culture +/- selection
for transposon integration. All cells are cultured at 37.degree. C.
after day 3. Cells cultured for 14+ days are collected for SURVEYOR
assay and cryopreserved for downstream applications, i.e.,
Single-Cell Nuclear Transfer. Panel b) Fibroblasts were transfected
using cationic-lipids. No activity was observed at day 3 (due to
low transfection efficiency) so only data for day 14+ populations.
Temperature treatment, selection and TALEN id (identified by
letters A-C as indicated in panel (c)) are shown above the gel.
Panel c) Fibroblasts were transfected by Nucleofection and percent
NHEJ was measured at day 3, and in day 14+ non-selected (NS) and
selected (S) populations. Temperature treatment is indicated above
each matrix. Abbreviations: nd=not detected; wt=wild type amplicon,
SURVEYOR treated.
[0022] FIG. 8A. Direct PCR sequencing for identification of indels.
PCR amplicons from individual fibroblast colonies were purified,
sequenced and compared to the wild-type sequence. Mutation of one
allele, or, non-overlapping mutations of both alleles will result
in double sequence near the TALEN recognition sites (top).
Overlapping bi-allelic mutations can be identified where
differences between each allele can be identified by double peaks
flanking the mutation site. Colonies with homozygous mutations do
not display double peaks near the indel site.
[0023] FIG. 8B. Sequence comparisons of wild-type and bi-allelic
clones with homozygous indels, as in FIG. 8A.
[0024] FIG. 9A. DMD Bi-allelic modification alleles. Colonies with
either homozygous modification alleles (i.e., both alleles harbor
the same mutation) or bi-allelic mutation with different mutations
on each allele are displayed. For colonies with two indels, the
number of times each allele was sequenced is displayed on the
right. In some cases, a third mutation or single wild-type allele
was sequenced, indicating that not all colonies are 100% clonal.
Frame-shift alleles are indicated and mismatch nucleotides are
denoted by lower-case text.
[0025] FIG. 9B. LDLR Bi-allelic modification alleles, with
notations as in FIG. 9A.
[0026] FIG. 10 TALEN-induced deletions and inversions. A schematic
of the DMD locus is shown in panel (a). DNA orientation is denoted
by black chevrons. TALENs targeted to exons 6 and 7 (black
arrowheads) co-transfected into male pig fibroblasts could result
in a NHEJ fusion event between exons 6 and 7. This could be
identified using primers (black arrows) resulting in .about.500 bp
amplicon. Panel b) SURVEYOR assay of cells transfected
simultaneously with TALENs targeted to exons 6 and 7 reveal NHEJ
indels at both sites. Percent NHEJ is displayed below. Panel c) PCR
with primers flanking the presumptive deletion site yield a
.about.500 basepair product when both exon-6 and exon-7 TALENs are
introduced simultaneously, but not when transfected singly. Panel
d) The predicted outcome of an inversion event of the sequence
between the TALEN target sites is shown. DNA orientation is denoted
by black chevrons. Primers outside the presumptive flanking sites
at the 5' and 3' end of the inversion locus are shown (black
arrows) along with predicted product size. PCR products were
observed at both 5' and 3' junctions only when both exon-6 and
exon-7 TALENs are introduced simultaneously.
[0027] FIG. 11 DMD deletion sequences. DMD deletion junctions from
replicate transfections are displayed. Above, exons 6 and 7
sequences are highlighted in green and yellow, respectively, and
TALEN-recognition sites are underlined. Inserted nucleotides are
highlighted in red.
[0028] FIG. 12 DMD inversion sequences. A schematic of the DMD
inversion allele is shown with the 5' and 3' junctions (boxed) that
were analyzed by sequencing. Below, the predicted sequence for each
fusion is shown corresponding fusion at the center of each spacer
for the TALEN pairs. TALEN-recognition sites are underlined.
Sequenced inversion alleles from a transfected population are
shown. The number of times each allele was sequenced is indicated
at the right and inserted nucleotides are underlined. Mismatched
nucleotides are denoted as lower-case text.
[0029] FIG. 13 HDR induction in bovine fibroblasts. Panel a) TALENs
(btGDF83.1, arrow) and a dsDNA template (BB-HDR) were designed to
introduce an 11-basepair deletion into exon-3 of bovine GDF8
(Belgium Blue mutation) by Double-Strand Break-induced homologous
recombination. Half of the binding site for the left TALEN is
missing in the BB-HDR template and thus should be resistant to
TALEN cleavage. Panel b) SURVEYOR assay demonstrates activity of
btGDF83.1 TALENs at both 37 and 30.degree. C. The PCR product used
for this assay was generated using primers b and b' (shown in panel
a). The BB-HDR template was not included in these replicates since
it would confound estimates of btGDF83.1 activity. Panel c)
Allele-specific PCR demonstrates that HDR induction is dependent on
co-transfection of TALENs and the BB-HDR template. The PCR assay
was developed to specifically detect HDR modified GDF8 alleles
using primers c and c' (shown panel a). The 3' end of primer c'
spans the 11-basepair deletion, and cannot amplify the wild type
allele (wt). Five hundred cell equivalents were included in each
PCR reaction including the positive control "C". Percent HDR was
determined by comparative densitometry between experimental and
control reactions.
[0030] FIG. 14 Confirmation of Belgian Blue introgression by
sequencing. The schematics of Wagyu wild-type GDF8 and the Belgian
Blue template (BB-HDR) are shown. PCR was conducted using primers
located outside of the homology arms (c and d) on five PCR positive
colonies followed by cloning and sequencing with primer b'.
Comparison to the wild-type sequence reveals the expected
11-basepair deletion characteristic the Belgian Blue allele
(heterozygous) in 4 of 5 colonies.
[0031] FIG. 15 Schematic and gel for TALEN-mediated HDR. A TALEN
pair (LDLR2.1) targeted to the fourth exon of the swine low density
lipoprotein receptor (LDLR) gene was co-transfected with the
supercoiled plasmid Ldlr-E4N-stop, which contains homology arms
corresponding to the swine LDLR gene and a gene-trap enabling
expression of Neomycin phosphotransferase upon HDR.
DETAILED DESCRIPTION
[0032] The successful use of TALENs to make genetic modifications
in higher animals such as swine or cows presents considerable
challenges. Compositions and methods of making such animals are set
forth herein. Some of these methods involve cloning from primary
artiodactyl cells, which also presents considerable challenges.
Further, methods for identifying cells or embryos that have been
modified with TALENs are presented, as well as processes for
enriching the percentage of TALEN-treated cells or embryos.
Unexpectedly, it was observed that a genetic modification of one
chromosome by a TALEN often caused the complementary locus of the
other chromosome to also be modified by cellular machinery.
[0033] Further, it was also discovered that TALENs could be used to
make gross chromosomal deletions (GCDs) at a plurality of sites.
FIG. 2 illustrates this approach, which involves a first TALEN pair
directed to a first locus and a second TALEN pair directed to a
second locus. It was also surprisingly discovered that inversions
of large chromosomal sequences could be created using pairs of
TALENs. One use of the inversions is the creation of artiodactyls
or other founder animals with fixed genetic traits, or the creation
of deletion strains.
Creation of Genetically Modified Livestock Via TALEN Technologies;
Verification of TALEN Modification; Co-Transfectional Co-Selection
for Selection of Modified Cells; Elimination of Reporter Genes from
Genetically Modified Livestock
[0034] One of the barriers to making TALEN-modified livestock is
that the efficiency of making a modification to an animal cell is
only a few percent with conventional best practices. Achievement of
a deletion or an insertion at an intended site does not necessarily
mean success because it may not actually create the intended
effect, such as expressing an exogenous protein or stopping
expression of an endogenous protein. Even a low efficiency can be
useful for the creation of genetically modified lower animals such
as fruit flies or mice because they have short and prolific
reproductive cycles that provide for the creating, testing, and
screening of hundreds of animals to determine if there are a few
that have been successfully modified. These levels of efficiency
that are conventionally achieved, however, are not suited to
livestock artiodactyls that have much longer gestational times and
comparatively few progeny per pregnancy.
[0035] Another barrier to using TALENs to modify livestock is that
TALEN-mediated modification of DNA in primary cells is difficult
because the cells are unstable. Indeed, it is shown herein that
frequency of TALEN-modified cells decreases significantly over time
in the absence of enrichment or selection methods. Without being
bound to a particular theory, it is theorized that DNA cleavage at
non-intended sites can compromise the stability of the cell by
inducing apoptosis or disabling non-target genes. The term primary
cell means a cell isolated from a living animal, wherein the cell
has undergone between 0 and 2 replications since its isolation from
the tissue.
[0036] As a result, techniques customarily used to create and test
transformed cells for successful genetic modification can not be
used in primary cells due to their propensity to senesce.
TALEN-modified cells are customarily destroyed to assay their
genetic modification, or isolated to grow clonal lines with many
identical cells from one parent. However, primary cells are
inherently unstable and typically undergo genetic changes,
senescence, and/or cell death when attempts are made to genetically
modify and clonally expand them. TALEN-modified cells are even less
stable, as documented herein for the first time. As a result, it is
unreasonable to expect high rates of success when using
conventional approaches that involve modifying a primary cell for
somatic cell nuclear transfer or other animal cloning technique. As
reported herein, however, TALENs have been used to make genetically
modified artiodactyl primary cells. These modifications are suited
to making founders of genetically modified animal lines by cloning.
Also described herein are direct-embryonic injections that were
used to modify zygotes, with the modified zygotes being suited to
implant into surrogate females for gestation and delivery of
founder animal lines.
[0037] A typical approach to testing for an actual TALEN-mediated
insertion/deletion event is to sequence the modified cell or
zygote, which is a destructive process. Thus when a zygote or
embryo is modified before implantation to a surrogate, its
modification can not be verified with any degree of convenience
until the animal is born. It is not conventionally appreciated that
an actual production process for making genetically modified
animals by cloning will benefit from a process for testing for the
presence of a genetic modification. There are inventions presented
herein that provide for an indication of genetic modification at
the single cell, zygote, or oocyte stage. As shown herein,
expression of a reporter gene that is not coupled to TALEN
modification is, despite not being part of the reporter gene
expression cassette, nonetheless generally predictive of a desired
genetic modification. More specifically, the expression of the
reporter gene indicates that the nucleic acids were effectively
delivered and being expressed in a cell or embryo; a
reporter-expressing cell or embryo is more likely to have undergone
TALEN-based modification.
[0038] Another technique for making modified organisms was the use
of a co-transfection, co-selection technique. The cells that
express the reporter are selected for, and may be used for making
genetically modified animals. The reporter may be chosen to require
transposase activity. Without being bound to a specific theory, it
is theorized that cells that have undergone transposition have 1)
been transfected and 2) been competent for double stranded DNA
repair, thus increasing the likelihood of TALEN-based modification
in selected clones. This also facilitates enrichment/selection for
transposed cells (and by extension TALEN-modified cells). The fact
that the transposon is operably but not physically linked to the
TALEN modification permits their segregation away from each other
by breeding. A benefit of a co-transfection strategy is that the
reporter, or reporters, may be placed on a chromosome that is not
the same chromosome that is modified by TALENs. This process
provides for the creation of founder animals that have no reporter
genes. For example, some animals were made by using plasmids
carrying reporter genes that were independent of the genetic
modification, which was orchestrated separately in the cells. This
scheme was based on a theory of operation that cells that
incorporate new reporter genes will also incorporate genetic
modifications. For instance, data provided herein shows that cells
can be transfected with four independent plasmids and the
successful incorporation of the gene product of one plasmid is
predictive of successful incorporation of the other plasmid gene
products and also for the success of TALEN-mediated changes.
[0039] TALEN function in livestock embryos was investigated using
in vitro prepared (IVP) bovine and porcine embryos. Example 1
describes direct injection of TALENs (a left TALEN and a right
TALEN) into bovine embryos to produce genetically modified animals
with a modification at the site where the TALENs specifically
bound. The modifications included homozygous-biallelic and
heterozygous-biallelic modifications. TALEN mRNAs were directly
injected into the embryos and successful genetic modifications were
observed. Example 2 describes additional experiments wherein
reporter mRNA was co-injected with TALEN mRNAs. Expression of the
reporter was predictive of a successful genetic modification, with
about 35% of the embryos expressing the reporter, and about 30% of
these animals having a TALEN-based indel. Of the animals with
indels, about 35% of them were either homozygous or heterozygous
bi-allelic mutants (FIG. 4). Direct embryo modification using
TALENs was thus shown to be a viable approach to livestock genome
modification. Embryos may thus be prepared and implanted into
surrogate females for gestation and delivery of animal founder
lines using well known processes. Moreover, it is possible to use a
reporter to select cells (e.g., primary cells, zygotes, oocytes,
blastocysts) for further use in cloning or other processes.
[0040] Methods for TALEN-mediated genetic modification of livestock
by cloning were also developed. Example 3 describes development of
suitable TALENs and TALEN modification of somatic primary cells of
swine and cows. The efficiency of successful modification was
somewhat low and no reporters for measuring success of the
modification were used. Nucleofection is a means for introducing
foreign nucleic acids into a cell with high efficiency, but it is
expensive, results in high levels of cytotoxicity, and is not
available to many researchers. Therefore, a common cationic lipid
transfection reagent was used as a vehicle for genetic
modification. As shown in Example 4, despite a less than 5%
transfection efficiency with cationic lipids, modification levels
were significantly enriched by transposon co-selection. Whereas
gene modification was below detection in day 3 populations (data
not shown) and day 14 populations without transposon-mediated
selection, modification levels in selected populations reached 31,
13 and 20 percent for DMD7.1, DMD6 and LDLR2.1 respectively (FIG.
7). Transposon co-selection was then applied to cells transfected
by nucleofection where >90% transfection efficiency is routine.
Transposon co-selection was effective for maintenance modified
cells transfected by Nucleofection, however, with the exception of
ACAN12, nucleofection did not significantly enrich for modified
cells over day 3 levels (FIG. 7). Thus, transposon co-selection is
an effective enrichment method when transfection efficiency is low
and an effective maintenance method when transfection efficiency is
high. Co-selection processes were also effective when feeder cells
were used, as demonstrated in Example 5. An unexpectedly high
proportion of bi-allelic modifications (about 17% to about 35%
depending on the TALEN-pair) were observed.
[0041] An embodiment of the invention is a composition and a method
for using TALENs to genetically modify livestock such as
artiodactyls. Many of the problems making these animals using
conventional processes have been discussed above. The genetic
modification may be, for example, chosen from the list consisting
of an insertion, a deletion, insertion of an exogenous nucleic acid
fragment, an inversion, and gene conversion to a natural or a novel
allele. For instance, an undesired mutation in a chromosome or
chromosome pair may be replaced with a normal sequence. In general,
a target DNA site is identified and a TALEN-pair is created that
will specifically bind to the site. The TALEN is delivered to the
cell or embryo, e.g., as a protein, mRNA or by a vector that
encodes the TALEN. The TALEN cleaves the DNA to make a
double-strand break that is then repaired, often resulting in the
creation of an indel, or incorporating sequences or polymorphisms
contained in an accompanying exogenous nucleic acid that is either
inserted or serves as a template for repair of the break with a
modified sequence. The term exogenous nucleic acid means a nucleic
acid that is added to the cell or embryo, regardless of whether the
nucleic acid is the same or distinct from nucleic acid sequences
naturally in the cell. The term nucleic acid fragment is broad and
includes a chromosome, expression cassette, gene, DNA, RNA, mRNA,
or portion thereof. The cell or embryo may be, for instance, chosen
from the group consisting of livestock, an artiodactyl, a cow, a
swine, a sheep, a goat, a chicken, a rabbit, and a fish. The term
livestock means domesticated animals that are raised as commodities
for food or biological material. The term artiodactyl means a
hoofed mammal of the order Artiodactyla, which includes cattle,
deer, camels, hippopotamuses, sheep, and goats that have an even
number of toes, usually two or sometimes four, on each foot.
[0042] One embodiment is directed to a composition or a method of
making a genetically modified livestock and/or artiodactyl
comprising introducing a TALEN-pair into livestock and/or an
artiodactyl cell or embryo that makes a genetic modification to DNA
of the cell or embryo at a site that is specifically bound by the
TALEN-pair, and producing the livestock animal/artiodactyl from the
cell. Direct injection may be used for the cell or embryo, e.g.,
into a zygote, blastocyst, or embryo. Alternatively, the TALEN
and/or other factors may be introduced into a cell using any of
many known techniques for introduction of proteins, RNA, mRNA, DNA,
or vectors. Genetically modified animals may be made from the
embryos or cells according to known processes, e.g., implantation
of the embryo into a gestational host, or various cloning methods.
The phrase "a genetic modification to DNA of the cell at a site
that is specifically bound by the TALEN", or the like, means that
the genetic modification is made at the site cut by the nuclease on
the TALEN when the TALEN is specifically bound to its target site.
The nuclease does not cut exactly where the TALEN-pair binds, but
rather at a defined site between the two binding sites.
[0043] Another such embodiment involves a composition or a
treatment of a cell that is used for cloning the animal. The cell
may be a livestock and/or artiodactyl cell, a cultured cell, a
primary cell, a primary somatic cell, a zygote, a germ cell, a
primordial germ cell, or a stem cell. For example, an embodiment is
a composition or a method of creating a genetic modification
comprising exposing a plurality of primary cells in a culture to
TALEN proteins or a nucleic acid encoding a TALEN or TALENs. The
TALENs may be introduced as proteins or as nucleic acid fragments,
e.g., encoded by mRNA or a DNA sequence in a vector.
[0044] Genetic modification of animals may also include
transfection with a reporter. As discussed above, primary cells
were observed to be unstable as a result of cellular modifications
mediated by the TALENs and/or TALENs introduction. As a result,
success in the modification of primary cells (among other cell
types), and/or the creation of new lines of livestock from such
cells is not reasonably expected using conventional means. It is
theorized, without being bound to a specific theory that cells that
express a gene cassette from a first vector are also likely to be
successfully modified by a TALEN delivered independently by mRNA or
another vector. Expression of a reporter allows for elimination of
cells that do not express the reporter. Alternatively, it allows
for moving cells that express the reporter from the culture for use
in animals by cloning or other transgenic animal techniques, or
into a second culture for further cultivation and/or expansion in
number and/or addition of further vectors and/or nucleic acids
and/or TALENs and/or other genetic modifications. The term
reporter, as used herein, includes reporters and selection
makers.
[0045] The term selection marker, as used herein, refers to a
genetically expressed biomolecule that confers a trait that permits
isolation by either positive or negative survival selection
criteria. The reporter may be, e.g., a florescent marker, e.g.,
green fluorescent protein and yellow fluorescent protein. The
reporter may be a selection marker, e.g., puromycin, ganciclovir,
adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo,
G418, APH), dihydrofolate reductase (DHFR),
hygromycin-B-phosphtransferase, thymidine kinase (TK), or
xanthin-guanine phosphoribosyltransferase (XGPRT).
[0046] Embodiments of the invention include introducing a reporter
(for instance by use of a vector) and a TALEN (e.g., by an
independent vector or mRNA) into a cell or embryo. The cell may be
a livestock and/or artiodactyl cell. The TALEN and/or reporter may
be directly introduced, e.g., by injection, or other means, e.g.,
involving cell culture. A cell culture may be made comprising
cultured cells (primary cells, zygotes, oocytes, immortalized
cells), a first nucleic acid encoding a TALEN, e.g., mRNA or a
vector with DNA encoding the TALEN, and an independent vector
having a DNA sequence encoding a reporter. The mRNA or first vector
do not encode any reporters and the second vector does not encode
any TALs and does not encode any TALENs
[0047] Vectors for the reporter, selection marker, and/or one or
more TALEN may be a plasmid, transposon, transposase, viral, or
other vectors, e.g., as detailed herein. Transposases may be used.
One embodiment involving a transposases provides a vector that
encodes a transposase. Other vectors encode a transposon that is
recognized by the transposase and has a nucleic acid fragment of
interest, e.g., a reporter, selection marker, exogenous nucleic
acid for insertion or as a template for modification, or one or
more TALENs. Accordingly, a cell or embryo may be transfected with
a number of vectors between, for example, 1 and about 6; artisans
will immediately appreciate that all the ranges and values within
the explicitly stated ranges are contemplated, e.g., 2, 3, 4, 5,
and 6. More vectors may be used. The reporter may be used to
identify cells that are likely to have undergone modification by
TALENs. Or a selection marker may be used to enrich the proportion
of TALEN-modified cells by destroying cells or embryos that do not
express the selection marker.
[0048] An embodiment of the invention is a cell or embryo culture
exposed to, or injected with, a plurality of vectors. A first
vector comprises a TALEN or TALEN-pair; alternatively there are two
TALEN vectors that independently provide a left TALEN and a right
TALEN. A second vector comprises a reporter. The reporter may
provide for non-destructive identification or may be a selection
marker. A vector encoding a selection marker may be used as an
alternative to the reporter vector, or in addition to the reporter
vector. A further vector may encode an exogenous nucleic acid.
[0049] A process for making TALEN-modified cells, embryos, or
animals comprises assaying a cell or embryo exposed to a TALEN for
expression of a reporter and using that cell or embryo in a method
or composition for making a genetically modified livestock and/or
artiodactyl. For instance, a primary cell may be removed from a
cell culture and used for cloning. Or, a primary cell may be
removed from culture and placed in a second culture to make a
clonal line or for further processes. Or, an embryo or zygote
expressing the reporter may be used for either implantation into a
surrogate dam or can be used for cloning, while other embryos or
zygotes that do not express the reporter not used for cloning. In
some embodiments, the reporter is a selection marker that is used
to select for cells or embryos that express the marker.
Gross Chromosomal Deletions and Inversions; Genetically Modified
Animals
[0050] Experiments were performed with TALENs directed to a
plurality of DNA sites. The sites were separated by several
thousand base pairs. It was observed that the DNA could be rejoined
with the deletion of the entire region between the sites.
Embodiments include, for example, sites separated by a distance
between 1-5 megabases or between 50% and 80% of a chromosome, or
between about 100 and about 1,000,000 basepairs; artisans will
immediately appreciate that all the ranges and values within the
explicitly stated ranges are contemplated, e.g., from about 1,000
to about 10,000 basepairs or from about 500 to about 500,000
basepairs. Alternatively, exogenous DNA may be added to the cell or
embryo for insertion of the exogenous DNA, or template-driven
repair of the DNA between the sites. Modification at a plurality of
sites may be used to make genetically modified cells, embryos,
artiodactyls, and livestock. Example 6 describes the deletion of
several thousand basepairs of DNA, with rejoining of the ends
verified biochemically.
[0051] Unexpectedly, TALEN-cleavage at separated sites also
resulted in frequent inversion of the entire region between TALEN
targets. As an additional surprise, as detailed in Example 6, these
inversions were accomplished with great fidelity. Forty one out of
43 of the tested inversions were positive at both the 5' and 3'
junctions. And sequencing of PCR products confirmed both deletion
and inversion events with addition or deletion of very few
nucleotides at their junctions (FIG. 11, 12). Cells or embryos with
these deletions or inversions have many uses as assay tools for
genetics.
[0052] These cells are also useful for making animals, livestock,
and animal models. Large deletions provide for gene inactivation.
Also, for instance, a deletion strain may be made of cells,
livestock, or animal models. Crossing the deletion strains with an
organism bearing a mutation for comparison to a wild-type helps to
rapidly and conveniently localize and identify the mutation locus.
Deletion strains are well known in these arts and involve sets of
organisms made with a series of deletions in a gene. Deletion
mapping involves crossing a strain which has a point mutation in a
gene with the deletion strains. Wherever recombination occurs
between the two strains to produce a wild-type (+) gene, the
mutation cannot lie within the region of the deletion. If
recombination cannot produce any wild-type genes, then it is
reasonable to conclude that the point mutation and deletion are
found within the same stretch of DNA. This can be used for example
to identify causative mutations, or to identify polymorphisms
underlying quantitative trait loci.
[0053] Cells, embryos, livestock, artiodactyls, and animal models
with inversions are also useful for fixing a genetic trait in
progeny of an organism or an animal line or animal breed.
Recombinations typically occur between homologous regions of
matching chromosomes during meiosis. Inversion of a chromosomal
region destroys homology and suppresses meiotic recombination.
Methods and compositions described herein may be used to make such
organisms or animals. For example, DNA in a somatic bovine or
porcine cell may be cut at a plurality of loci by TALENs, and cells
with an inversion may be isolated, or cells expressing reporters
may be used as likely candidates for successful inversions. The
cells may be used to clone animals that harbor chromosomal regions
that are incapable of meiotic recombinations. Alternatively, it is
expected that inversions will also occur at reasonable frequencies
in embryos treated with multiple TALEN-pairs at plurality of
sites.
[0054] An embodiment of this method is identifying a DNA region
encoding a genetic trait and cutting a DNA in a cell or embryo on
each side of the encoded trait at sites using a plurality of
TALENs. The modified cell or embryo may be used for creating a
genetically modified animal. The method may comprise isolating a
genetically modified animal that has the inversion.
Movement of Alleles
[0055] Some livestock traits are related to alleles such as
polymorphisms (large or small), single nucleotide polymorphisms,
deletions, insertions, or other variations. For instance, a
myostatin allele (an 11-bp deletion) from Belgian Blue cattle is
well known to cause a double-muscling phenotype. Example 7 shows,
using the Belgian Blue allele, how to precisely transfer specific
alleles from one livestock breed to another by homology-dependent
repair (HDR). Bovine fibroblasts received the allele and may
readily be used to make transgenic cattle. This allele does not
interfere with normal development and the methods taught herein
place the allele with precision and without disruption of other
genes or the incorporation of exogenous genes. As already
discussed, results presented herein show that the frequency of
allele conversion in livestock fibroblasts is high when sister
chromatids are used for an HDR template, therefore allele
introgression into one sister chromatid can be anticipated
frequently to result in homozygosity.
[0056] An embodiment of this invention is a method of transfer of
an allele from a first livestock line or breed to a second
livestock line or breed, comprising cutting DNA with a pair of
TALENs in a cell or embryo of the second livestock line/breed in a
presence of a nucleic acid that encodes the allele of the first
livestock line/breed. The embryo or cell may be used to create an
animal of the second line/breed that has the allele of the first
line/breed. The DNA that encodes the allele provides a template for
homology-dependent repair. As a template, it has homology to
portions of the DNA on each side of the cut and also contains the
desired allele. Embodiments of the invention comprise moving a
plurality of alleles from one breed to another breed. As set forth
elsewhere herein, the TALENs may be delivered a protein or encoded
by a nucleic acid, e.g., an mRNA or a vector. A reporter may also
be transfected into the cell or embryo and used as a basis for
selecting TALEN-modified cells. The reporter may be assayed
non-destructively and/or may comprise a selection marker. The term
breed means a group of domestic animals or plants with a
homogeneous appearance, behavior, and other characteristics that
distinguish it from other animals or plants of the same species.
The animals that belong to a particular breed are known to artisans
that practice in these arts.
[0057] A population or species of organisms typically includes
multiple alleles at each locus among various individuals. Allelic
variation at a locus is measurable as the number of alleles
(polymorphisms) present, or the proportion of heterozygotes in the
population. For example, at the gene locus for the ABO blood type
carbohydrate antigens in humans, classical genetics recognizes
three alleles, IA, IB, and IO that determine compatibility of blood
transfusions. An allele is a term that means one of two or more
forms of a gene.
[0058] In livestock, many alleles are known to be linked to various
traits such as production traits, type traits, workability traits,
and other functional traits. Artisans are accustomed to monitoring
and quantifying these traits, e.g., Visscher et al., Livestock
Production Science, 40 (1994) 123-137, U.S. Pat. No. 7,709,206, US
2001/0016315, US 2011/0023140, and US 2005/0153317. Accordingly,
the allele that is transferred may be linked to a trait or chosen
from a trait in the group consisting of a production trait, a type
trait, a workability trait, a fertility trait, a mothering trait,
and a disease resistance trait.
[0059] The term natural allele in the context of genetic
modification means an allele found in nature in the same species of
organism that is being modified. The term novel allele means a
non-natural allele. A human allele placed into a goat is a novel
allele. The term synthetic allele means an allele that is not found
in nature. Thus a natural allele is a variation already existing
within a species that can be interbred. And a novel allele is one
that does not exist within a species that can be interbred.
[0060] Moving an allele from one breed to another by conventional
breeding processes involves swapping many alleles between the
breeds. Recombination during meiosis inevitably exchanges genetic
loci between the breeds. In contrast, the TALENs-modified livestock
and other animals are free of meiotic recombination events. As a
result, a TALEN-modified animal can easily be distinguished from an
animal created by sexual reproduction.
Compositions and Kits
[0061] The present invention also provides compositions and kits
containing, for example, nucleic acid molecules encoding TALENs,
TALEN polypeptides, compositions containing such nucleic acid
molecules or polypeptides, or TALEN engineered cell lines. Such
items can be used, for example, as research tools, or
therapeutically.
Recombinases
[0062] Embodiments of the invention include administration of a
TALEN or TALENs with a recombinase. A recombinase forms a filament
with a nucleic acid fragment and, in effect, searches cellular DNA
to find a DNA sequence substantially homologous to the sequence. An
embodiment of a TALEN-recombinase embodiment comprises combining a
recombinase with a nucleic acid sequence that serves as a template
for HDR. The HDR template sequence has substantial homology to a
site that is targeted for cutting by the TALEN/TALEN pair. As
described herein, the HDR template provides for a change to the
native DNA, by placement of an allele, creation of an indel,
insertion of exogenous DNA, or with other changes. The TALEN is
placed in the cell or embryo by methods described herein as a
protein, mRNA, or by use of a vector. The recombinase is combined
with the HDR to form a filament and placed into the cell. The
recombinase and/or HDR template that combines with the recombinase
may be placed in the cell or embryo as a protein, an mRNA, or with
a vector that encodes the recombinase. The disclosure of US Pub
2011/0059160 (U.S. Ser. No. 12/869,232) is hereby incorporated
herein by reference for all purposes; in case of conflict, the
specification is controlling. The term recombinase refers to a
genetic recombination enzyme that enzymatically catalyzes, in a
cell, the joining of relatively short pieces of DNA between two
relatively longer DNA strands. Recombinases include Cre
recombinase, Hin recombinase, RecA, RAD51, Cre, and FLP. Cre
recombinase is a Type I topoisomerase from P1 bacteriophage that
catalyzes site-specific recombination of DNA between loxP sites.
Hin recombinase is a 21 kD protein composed of 198 amino acids that
is found in the bacteria Salmonella. Hin belongs to the serine
recombinase family of DNA invertases in which it relies on the
active site serine to initiate DNA cleavage and recombination.
RAD51 is a human gene. The protein encoded by this gene is a member
of the RAD51 protein family which assist in repair of DNA double
strand breaks. RAD51 family members are homologous to the bacterial
RecA and yeast Rad51. Cre recombinase is an experimental enzyme
that in lab tests has successfully removed DNA inserted by HIV from
infected cells. The enzyme was derived from Cre recombinase through
selective mutation for the purposes of identifying HIV markers,
which are not bounded by loxP sites and therefore disallow attempts
at Cre-Lox recombination. FLP refers to Flippase recombination
enzyme (FLP or Flp) derived from the 2.mu. plasmid of the baker's
yeast Saccharomyces cerevisiae.
[0063] RecA is known for its recombinase activity to catalyze
strand exchange during the repair of double-strand breaks by
homologous recombination (McGrew and Knight, 2003) Radding, et al.,
1981; Seitz et al., 1998). RecA has also been shown to catalyze
proteolysis, e.g., of the LexA and .lamda. repressor proteins, and
to possess DNA-dependent ATPase activity. After a double-strand
break occurs from ionizing radiation or some other insult,
exonucleases chew back the DNA ends 5' to 3', thereby exposing one
strand of the DNA (Cox, 1999; McGrew and Knight, 2003). The
single-stranded DNA becomes stabilized by single-strand binding
protein (SSB). After binding of SSB, RecA binds the single-stranded
(ss) DNA and forms a helical nucleoprotein filament (referred to as
a filament or a presynaptic filament). During DNA repair, the
homology-searching functions of RecA direct the filament to
homologous DNA and catalyze homologous base pairing and strand
exchange. This results in the formation of DNA heteroduplex. After
strand invasion, DNA polymerase elongates the ssDNA based on the
homologous DNA template to repair the DNA break, and crossover
structures or Holliday junctions are formed. RecA also shows a
motor function that participates in the migration of the crossover
structures (Campbell and Davis, 1999).
[0064] Recombinase activity comprises a number of different
functions. For example, polypeptide sequences having recombinase
activity are able to bind in a non-sequence-specific fashion to
single-stranded DNA to form a nucleoprotein filament. Such
recombinase-bound nucleoprotein filaments are able to interact in a
non-sequence-specific manner with a double-stranded DNA molecule,
search for sequences in the double-stranded molecule that are
homologous to sequences in the filament, and, when such sequences
are found, displace one of the strands of the double-stranded
molecule to allow base-pairing between sequences in the filament
and complementary sequences in one of the strands of the double
stranded molecule. Such steps are collectively denoted
"synapsis."
[0065] RecA and RecA-like proteins (called Rad51 in many eukaryotic
species) have been examined for stimulating gene targeting and
homologous recombination in a variety of eukaryotic systems. In
tobacco cells, expression of bacterial RecA containing a nuclear
localization signal (NLS) increases the repair of mitomycin
C-induced DNA damage by homologous recombination and somatic
intrachromosomal recombination (recombination between homologous
chromosomes) from three to ten fold (Reiss et al., 1996).
Expression of NLSRecA in tobacco can also stimulate sister
chromatid exchange 2.4-fold over wild-type levels (Reiss et al.,
2000). In somatic mammalian cells, overexpression of NLSRecA
stimulates gene-targeting by homologous recombination 10-fold
(Shcherbakova et al., 2000). However, in human cells,
overexpression of a human homologue of RecA, hRAD51, only
stimulates recombination 2 to 3-fold over wild type levels under
the antibiotic selection (Yanez and Porter, 1999). In zebrafish, a
mutant form of the enhanced green fluorescent protein (EGFP) was
corrected at low frequency by injecting ssDNA-RecA filaments
directly (Cui et al., 2003). Rad52, a member of the Rad51 epistasis
group, also promotes single-strand annealing and low level gene
disruption in zebrafish using mutated oligonucleotides (Takahashi
and Dawid, 2005). Taken together, these studies indicate that
ectopic expression of RecA or Rad51 results in a modest stimulation
of homologous recombination but does not increase levels
sufficiently to be useful for gene-targeting.
[0066] Thus recombinase activities include, but are not limited to,
single-stranded DNA-binding, synapsis, homology searching, duplex
invasion by single-stranded DNA, heteroduplex formation, ATP
hydrolysis and proteolysis. The prototypical recombinase is the
RecA protein from E. coli. See, for example, U.S. Pat. No.
4,888,274. Prokaryotic RecA-like proteins have also been described
in Salmonella, Bacillus and Proteus species. A thermostable RecA
protein, from Thermus aquaticus, has been described in U.S. Pat.
No. 5,510,473. A bacteriophage T4 homologue of RecA, the UvsX
protein, has been described. RecA mutants, having altered
recombinase activities, have been described, for example, in U.S.
Pat. Nos. 6,774,213; 7,176,007 and 7,294,494. Plant RecA homologues
are described in, for example, U.S. Pat. Nos. 5,674,992; 6,388,169
and 6,809,183. RecA fragments containing recombinase activity have
been described, for example, in U.S. Pat. No. 5,731,411. Mutant
RecA proteins having enhanced recombinase activity such as, for
example, RecA803 have been described. See, for example, Madiraju et
al. (1988) Proc. Natl. Acad. Sci. USA 85:6592-6596.
[0067] A eukaryotic homologue of RecA, also possessing recombinase
activity, is the Rad51 protein, first identified in the yeast
Saccharomyces cerevisiae. See Bishop et al., (1992) Cell 69:
439-56; Shinohara et al, (1992) Cell: 457-70; Aboussekhra, et al.,
(1992) Mol. Cell. Biol. 72, 3224-3234 and Basile et al., (1992)
Mol. Cell. Biol. 12, 3235-3246. Plant Rad51 sequences are described
in U.S. Pat. Nos. 6,541,684; 6,720,478; 6,905,857 and 7,034,117.
Another yeast protein that is homologous to RecA is the Dmc1
protein. RecA/Rad51 homologues in organisms other than E. coli and
S. cerevisiae have been described. Morita et al. (1993) Proc. Natl.
Acad. Sci. USA 90:6577-6580; Shinohara et al. (1993) Nature Genet.
4:239-243; Heyer (1994) Experientia 50:223-233; Maeshima et al.
(1995) Gene 160:195-200; U.S. Pat. Nos. 6,541,684 and
6,905,857.
[0068] Herein, "RecA" or "RecA protein" refers to a family of
RecA-like recombination proteins having essentially all or most of
the same functions, particularly: (i) the ability to position
properly oligonucleotides or polynucleotides on their homologous
targets for subsequent extension by DNA polymerases; (ii) the
ability topologically to prepare duplex nucleic acid for DNA
synthesis; and, (iii) the ability of RecA/oligonucleotide or
RecA/polynucleotide complexes efficiently to find and bind to
complementary sequences. The best characterized RecA protein is
from E. coli; in addition to the original allelic form of the
protein a number of mutant RecA-like proteins have been identified,
for example, RecA803. Further, many organisms have RecA-like
strand-transfer proteins including, for example, yeast, Drosophila,
mammals including humans, and plants. These proteins include, for
example, Rec1, Rec2, Rad51, Rad51B, Rad51C, Rad51D, Rad51E, XRCC2
and DMC1. An embodiment of the recombination protein is the RecA
protein of E. coli. Alternatively, the RecA protein can be the
mutant RecA-803 protein of E. coli, a RecA protein from another
bacterial source or a homologous recombination protein from another
organism.
[0069] Additional descriptions of proteins having recombinase
activity are found, for example, in Fugisawa et al. (1985) Nucl.
Acids Res. 13:7473; Hsieh et al. (1986) Cell 44:885; Hsieh et al.
(1989) J. Biol. Chem. 264:5089; Fishel et al. (1988) Proc. Natl.
Acad. Sci. USA 85:3683; Cassuto et al. (1987) Mol. Gen. Genet.
208:10; Ganea et al. (1987) Mol. Cell. Biol. 7:3124; Moore et al.
(1990) J. Biol. Chem.: 11108; Keene et al. (1984) Nucl. Acids Res.
12:3057; Kimiec (1984) Cold Spring Harbor Symp. 48:675; Kimeic
(1986) Cell 44:545; Kolodner et al. (1987) Proc. Natl. Acad. Sci.
USA 84:5560; Sugino et al. (1985) Proc. Natl. Acad, Sci. USA 85:
3683; Halbrook et al. (1989) J. Biol. Chem. 264:21403; Eisen et al.
(1988) Proc. Natl. Acad. Sci. USA 85:7481; McCarthy et al. (1988)
Proc. Natl. Acad. Sci. USA 85:5854; and Lowenhaupt et al. (1989) J.
Biol. Chem. 264:20568, which are incorporated herein by reference.
See also Brendel et al. (1997) J. Mol. Evol. 44:528.
[0070] Examples of proteins having recombinase activity include
recA, recA803, uvsX, and other recA mutants and recA-like
recombinases (Roca (1990) Crit. Rev. Biochem. Molec. Biol. 25:415),
(Kolodner et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84:5560;
Tishkoff et al. (1991) Molec. Cell. Biol. 11:2593), RuvC
(Dunderdale et al. (1991) Nature 354:506), DST2, KEM1 and XRN1
(Dykstra et al. (1991) Molec. Cell. Biol. 11:2583), STPa/DST1
(Clark et al. (1991) Molec. Cell. Biol. 11:2576), HPP-1 (Moore et
al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:9067), other eukaryotic
recombinases (Bishop et al. (1992) Cell 69:439; and Shinohara et
al. (1992) Cell 69:457); incorporated herein by reference.
[0071] In vitro-evolved proteins having recombinase activity have
been described in U.S. Pat. No. 6,686,515. Further publications
relating to recombinases include, for example, U.S. Pat. Nos.
7,732,585, 7,361,641, 7,144,734. For a review of recombinases, see
Cox (2001) Proc. Natl. Acad. Sci. USA 98:8173-8180.
[0072] A nucleoprotein filament, or "filament" may be formed. The
term filament, in the context of forming a structure with a
recombinase, is a term known to artisans in these fields. The
nucleoprotein filament so formed can then be, e.g., contacted with
another nucleic acid or introduced into a cell. Methods for forming
nucleoprotein filaments, wherein the filaments comprise polypeptide
sequences having recombinase activity and a nucleic acid, are
well-known in the art. See, e.g., Cui et al. (2003) Marine
Biotechnol. 5:174-184 and U.S. Pat. Nos. 4,888,274; 5,763,240;
5,948,653 and 7,199,281, the disclosures of which are incorporated
by reference for the purposes of disclosing exemplary techniques
for binding recombinases to nucleic acids to form nucleoprotein
filaments.
[0073] In general, a molecule having recombinase activity is
contacted with a linear, single-stranded nucleic acid. The linear,
single-stranded nucleic acid may be a probe. The methods of
preparation of such single stranded nucleic acids are known. The
reaction mixture typically contains a magnesium ion. Optionally,
the reaction mixture is buffered and optionally also contains ATP,
dATP or a nonhydrolyzable ATP analogue, such as, for example,
.gamma.-thio-ATP (ATP-.gamma.-S) or .gamma.-thio-GTP
(GTP-.gamma.-S). Reaction mixtures can also optionally contain an
ATP-generating system. Double-stranded DNA molecules can be
denatured (e.g., by heat or alkali) either prior to, or during,
filament formation. Optimization of the molar ratio of recombinase
to nucleic acid is within the skill of the art. For example, a
series of different concentrations of recombinase can be added to a
constant amount of nucleic acid, and filament formation assayed by
mobility in an agarose or acrylamide gel. Because bound protein
retards the electrophoretic mobility of a polynucleotide, filament
formation is evidenced by retarded mobility of the nucleic acid.
Either maximum degree of retardation, or maximum amount of nucleic
acid migrating with a retarded mobility, can be used to indicate
optimal recombinase: nucleic acid ratios. Protein-DNA association
can also be quantitated by measuring the ability of a
polynucleotide to bind to nitrocellulose.
TALENs
[0074] The term TALEN, as used herein, is broad and includes a
monomeric TALEN that can cleave double stranded DNA without
assistance from another TALEN. The term TALEN is also used to refer
to one or both members of a pair of TALENs that are engineered to
work together to cleave DNA at the same site. TALENs that work
together may be referred to as a left-TALEN and a right-TALEN,
which references the handedness of DNA.
[0075] Miller et al. (Miller et al. (2011) Nature Biotechnol
29:143) reported making TALENs for site-specific nuclease
architecture by linking TAL truncation variants to the catalytic
domain of FokI nuclease. The resulting TALENs were shown to induce
gene modification in immortalized human cells by means of the two
major eukaryotic DNA repair pathways, non-homologous end joining
(NHEJ) and homology directed repair. The TALENs can be engineered
for specific binding. Specific binding, as that term is commonly
used in the biological arts, refers to a molecule that binds to a
target with a relatively high affinity compared to non-target
tissues, and generally involves a plurality of non-covalent
interactions, such as electrostatic interactions, van der Waals
interactions, hydrogen bonding, and the like. Specific binding
interactions characterize antibody-antigen binding,
enzyme-substrate binding, and specifically binding protein-receptor
interactions.
[0076] The cipher for TALs has been reported (PCT Application WO
2011/072246) wherein each DNA binding repeat is responsible for
recognizing one base pair in the target DNA sequence. The residues
may be assembled to target a DNA sequence, with: (a) HD for
recognition of C/G; (b) NI for recognition of A/T; (c) NG for
recognition of T/A; (d) NS for recognition of C/G or A/T or T/A or
G/C; (e) NN for 30 recognition of G/C or A/T; (f) IG for
recognition of T/A; (g) N for recognition of C/G; (h) HG for
recognition of C/G or T/A; (i) H for recognition of T/A; and (j) NK
for recognition of G/C. In brief, a target site for binding of a
TALEN is determined and a fusion molecule comprising a nuclease and
a series of RVDs that recognize the target site is created. Upon
binding, the nuclease cleaves the DNA so that cellular repair
machinery can operate to make a genetic modification at the cut
ends. The term TALEN means a protein comprising a Transcription
Activator-like (TAL) effector binding domain and a nuclease domain
and includes monomeric TALENs that are functional per se as well as
others that require dimerization with another monomeric TALEN. The
dimerization can result in a homodimeric TALEN when both monomeric
TALEN are identical or can result in a heterodimeric TALEN when
monomeric TALEN are different.
[0077] In some embodiments, a monomeric TALEN can be used. TALEN
typically function as dimers across a bipartite recognition site
with a spacer, such that two TAL effector domains are each fused to
a catalytic domain of the FokI restriction enzyme, the
DNA-recognition sites for each resulting TALEN are separated by a
spacer sequence, and binding of each TALEN monomer to the
recognition site allows FokI to dimerize and create a double-strand
break within the spacer. Monomeric TALENs also can be constructed,
however, such that single TAL effectors are fused to a nuclease
that does not require dimerization to function. One such nuclease,
for example, is a single-chain variant of FokI in which the two
monomers are expressed as a single polypeptide. Other naturally
occurring or engineered monomeric nucleases also can serve this
role. The DNA recognition domain used for a monomeric TALEN can be
derived from a naturally occurring TAL effector. Alternatively, the
DNA recognition domain can be engineered to recognize a specific
DNA target. Engineered single-chain TALENs may be easier to
construct and deploy, as they require only one engineered DNA
recognition domain. A dimeric DNA sequence-specific nuclease can be
generated using two different DNA binding domains (e.g., one TAL
effector binding domain and one binding domain from another type of
molecule). TALENs may function as dimers across a bipartite
recognition site with a spacer. This nuclease architecture also can
be used for target-specific nucleases generated from, for example,
one TALEN monomer and one zinc finger nuclease monomer. In such
cases, the DNA recognition sites for the TALEN and zinc finger
nuclease monomers can be separated by a spacer of appropriate
length. Binding of the two monomers can allow FokI to dimerize and
create a double-strand break within the spacer sequence. DNA
binding domains other than zinc fingers, such as homeodomains, myb
repeats or leucine zippers, also can be fused to FokI and serve as
a partner with a TALEN monomer to create a functional nuclease.
[0078] In some embodiments, a TAL effector can be used to target
other protein domains (e.g., non-nuclease protein domains) to
specific nucleotide sequences. For example, a TAL effector can be
linked to a protein domain from, without limitation, a DNA 20
interacting enzyme (e.g., a methylase, a topoisomerase, an
integrase, a transposase, or a ligase), a transcription activators
or repressor, or a protein that interacts with or modifies other
proteins such as histones. Applications of such TAL effector
fusions include, for example, creating or modifying epigenetic
regulatory elements, making site-specific insertions, deletions, or
repairs in DNA, controlling gene expression, and modifying
chromatin structure.
[0079] The spacer of the target sequence can be selected or varied
to modulate TALEN specificity and activity. The flexibility in
spacer length indicates that spacer length can be chosen to target
particular sequences with high specificity. Further, the variation
in activity has been observed for different spacer lengths
indicating that spacer length can be chosen to achieve a desired
level of TALEN activity.
[0080] The term nuclease includes exonucleases and endonucleases.
The term endonuclease refers to any wild-type or variant enzyme
capable of catalyzing the hydrolysis (cleavage) of bonds between
nucleic acids within a DNA or RNA molecule, preferably a DNA
molecule. Non-limiting examples of endonucleases include type II
restriction endonucleases such as FokI, HhaI, HindIII, NotI, BbvCl,
EcoRI, BglII, and AlwI. Endonucleases comprise also rare-cutting
endonucleases when having typically a polynucleotide recognition
site of about 12-45 basepairs (bp) in length, more preferably of
14-45 bp. Rare-cutting endonucleases induce DNA double-strand
breaks (DSBs) at a defined locus. Rare-cutting endonucleases can
for example be a homing endonuclease, a chimeric Zinc-Finger
nuclease (ZFN) resulting from the fusion of engineered zinc-finger
domains with the catalytic domain of a restriction enzyme such as
FokI or a chemical endonuclease. In chemical endonucleases, a
chemical or peptidic cleaver is conjugated either to a polymer of
nucleic acids or to another DNA recognizing a specific target
sequence, thereby targeting the cleavage activity to a specific
sequence. Chemical endonucleases also encompass synthetic nucleases
like conjugates of orthophenanthroline, a DNA cleaving molecule,
and triplex-forming oligonucleotides (TFOs), known to bind specific
DNA sequences. Such chemical endonucleases are comprised in the
term "endonuclease" according to the present invention. Examples of
such endonuclease include I-See I, I-Chu L I-Cre I, I-Csm I, PI-See
L PI-Tti L PI-Mtu I, I-Ceu I, I-See IL 1-See III, HO, PI-Civ I,
PI-Ctr L PI-Aae I, PI-Bsu I, PI-Dha I, PI-Dra L PI-Mav L PI-Meh I,
PI-Mfu L PI-Mfl I, PI-Mga L PI-Mgo I, PI-Min L PI-Mka L PI-Mle I,
PI-Mma I, PI-30 Msh L PI-Msm I, PI-Mth I, PI-Mtu I, PI-Mxe I,
PI-Npu I, PI-Pfu L PI-Rma I, PI-Spb I, PI-Ssp L PI-Fae L PI-Mja I,
PI-Pho L PI-Tag L PI-Thy I, PI-Tko I, PI-Tsp I, I-MsoI.
Genetically Modified Animals
[0081] Various techniques known in the art can be used to introduce
nucleic acid constructs into non-human animals to produce founder
lines, in which the nucleic acid construct is integrated into the
genome. Such techniques include, without limitation, pronuclear
microinjection (U.S. Pat. No. 4,873,191), retrovirus mediated gene
transfer into germ lines (Van der Putten et al. (1985) Proc. Natl.
Acad. Sci. USA 82, 6148-1652), gene targeting into embryonic stem
cells (Thompson et al. (1989) Cell 56, 313-321), electroporation of
embryos (Lo (1983) Mol. Cell. Biol. 3, 1803-1814), sperm-mediated
gene transfer (Lavitrano et al. (2002) Proc. Natl. Acad. Sci. USA
99, 14230-14235; Lavitrano et al. (2006) Reprod. Fert. Develop. 18,
19-23), and in vitro transformation of somatic cells, such as
cumulus or mammary cells, or adult, fetal, or embryonic stem cells,
followed by nuclear transplantation (Wilmut et al. (1997) Nature
385, 810-813; and Wakayama et al. (1998) Nature 394, 369-374).
Pronuclear microinjection, sperm mediated gene transfer, and
somatic cell nuclear transfer are particularly useful
techniques.
[0082] Typically, in pronuclear microinjection, a nucleic acid
construct is introduced into a fertilized egg; 1 or 2 cell
fertilized eggs are used as the pronuclei containing the genetic
material from the sperm head and the egg are visible within the
protoplasm. Pronuclear staged fertilized eggs can be obtained in
vitro or in vivo (i.e., surgically recovered from the oviduct of
donor animals). In vitro fertilized eggs can be produced as
follows. For example, swine ovaries can be collected at an
abattoir, and maintained at 22-28.degree. C. during transport.
Ovaries can be washed and isolated for follicular aspiration, and
follicles ranging from 4-8 mm can be aspirated into 50 mL conical
centrifuge tubes using 18 gauge needles and under vacuum.
Follicular fluid and aspirated oocytes can be rinsed through
pre-filters with commercial TL-HEPES (Minitube, Verona, Wis.).
Oocytes surrounded by a compact cumulus mass can be selected and
placed into TCM-199 OOCYTE MATURATION MEDIUM (Minitube, Verona,
Wis.) supplemented with 0.1 mg/mL cysteine, 10 ng/mL epidermal
growth factor, 10% porcine follicular fluid, 50 .mu.M
2-mercaptoethanol, 0.5 mg/ml cAMP, 10 IU/mL each of pregnant mare
serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG)
for approximately 22 hours in humidified air at 38.7.degree. C. and
5% CO.sub.2. Subsequently, the oocytes can be moved to fresh
TCM-199 maturation medium, which will not contain cAMP, PMSG or hCG
and incubated for an additional 22 hours. Matured oocytes can be
stripped of their cumulus cells by vortexing in 0.1% hyaluronidase
for 1 minute.
[0083] For swine, mature oocytes can be fertilized in 500 .mu.l
Minitube PORCPRO IVF MEDIUM SYSTEM (Minitube, Verona, Wis.) in
Minitube 5-well fertilization dishes. In preparation for in vitro
fertilization (IVF), freshly-collected or frozen boar semen can be
washed and resuspended in PORCPRO IVF Medium to 4.times.10.sup.5
sperm. Sperm concentrations can be analyzed by computer assisted
semen analysis (SPERMVISION, Minitube, Verona, Wis.). Final in
vitro insemination can be performed in a 10 .mu.l volume at a final
concentration of approximately 40 motile sperm/oocyte, depending on
boar. Incubate all fertilizing oocytes at 38.7.degree. C. in 5.0%
CO.sub.2 atmosphere for 6 hours. Six hours post-insemination,
presumptive zygotes can be washed twice in NCSU-23 and moved to 0.5
mL of the same medium. This system can produce 20-30% blastocysts
routinely across most boars with a 10-30% polyspermic insemination
rate.
[0084] Linearized nucleic acid constructs can be injected into one
of the pronuclei. Then the injected eggs can be transferred to a
recipient female (e.g., into the oviducts of a recipient female)
and allowed to develop in the recipient female to produce the
transgenic animals. In particular, in vitro fertilized embryos can
be centrifuged at 15,000.times.g for 5 minutes to sediment lipids
allowing visualization of the pronucleus. The embryos can be
injected with using an Eppendorf FEMTOJET injector and can be
cultured until blastocyst formation. Rates of embryo cleavage and
blastocyst formation and quality can be recorded.
[0085] Embryos can be surgically transferred into uteri of
asynchronous recipients. Typically, 100-200 (e.g., 150-200) embryos
can be deposited into the ampulla-isthmus junction of the oviduct
using a 5.5-inch TOMCAT.RTM. catheter. After surgery, real-time
ultrasound examination of pregnancy can be performed.
[0086] In somatic cell nuclear transfer, a transgenic artiodactyl
cell (e.g., a transgenic pig cell or bovine cell) such as an
embryonic blastomere, fetal fibroblast, adult ear fibroblast, or
granulosa cell that includes a nucleic acid construct described
above, can be introduced into an enucleated oocyte to establish a
combined cell. Oocytes can be enucleated by partial zona dissection
near the polar body and then pressing out cytoplasm at the
dissection area. Typically, an injection pipette with a sharp
beveled tip is used to inject the transgenic cell into an
enucleated oocyte arrested at meiosis 2. In some conventions,
oocytes arrested at meiosis-2 are termed "eggs." After producing a
porcine or bovine embryo (e.g., by fusing and activating the
oocyte), the embryo is transferred to the oviducts of a recipient
female, about 20 to 24 hours after activation. See, for example,
Cibelli et al. (1998) Science 280, 1256-1258 and U.S. Pat. No.
6,548,741. For pigs, recipient females can be checked for pregnancy
approximately 20-21 days after transfer of the embryos.
[0087] Standard breeding techniques can be used to create animals
that are homozygous for the target nucleic acid from the initial
heterozygous founder animals. Homozygosity may not be required,
however. Transgenic pigs described herein can be bred with other
pigs of interest.
[0088] In some embodiments, a nucleic acid of interest and a
selectable marker can be provided on separate transposons and
provided to either embryos or cells in unequal amount, where the
amount of transposon containing the selectable marker far exceeds
(5-10 fold excess) the transposon containing the nucleic acid of
interest. Transgenic cells or animals expressing the nucleic acid
of interest can be isolated based on presence and expression of the
selectable marker. Because the transposons will integrate into the
genome in a precise and unlinked way (independent transposition
events), the nucleic acid of interest and the selectable marker are
not genetically linked and can easily be separated by genetic
segregation through standard breeding. Thus, transgenic animals can
be produced that are not constrained to retain selectable markers
in subsequent generations, an issue of some concern from a public
safety perspective.
[0089] Once transgenic animal have been generated, expression of a
target nucleic acid can be assessed using standard techniques.
Initial screening can be accomplished by Southern blot analysis to
determine whether or not integration of the construct has taken
place. For a description of Southern analysis, see sections
9.37-9.52 of Sambrook et al., 1989, Molecular Cloning, A Laboratory
Manual, second edition, Cold Spring Harbor Press, Plainview; NY.
Polymerase chain reaction (PCR) techniques also can be used in the
initial screening. PCR refers to a procedure or technique in which
target nucleic acids are amplified. Generally, sequence information
from the ends of the region of interest or beyond is employed to
design oligonucleotide primers that are identical or similar in
sequence to opposite strands of the template to be amplified. PCR
can be used to amplify specific sequences from DNA as well as RNA,
including sequences from total genomic DNA or total cellular RNA.
Primers typically are 14 to 40 nucleotides in length, but can range
from 10 nucleotides to hundreds of nucleotides in length. PCR is
described in, for example PCR Primer: A Laboratory Manual, ed.
Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press,
1995. Nucleic acids also can be amplified by ligase chain reaction,
strand displacement amplification, self-sustained sequence
replication, or nucleic acid sequence-based amplified. See, for
example, Lewis (1992) Genetic Engineering News 12, 1; Guatelli et
al. (1990) Proc. Natl. Acad. Sci. USA 87:1874; and Weiss (1991)
Science 254:1292. At the blastocyst stage, embryos can be
individually processed for analysis by PCR, Southern hybridization
and splinkerette PCR (see, e.g., Dupuy et al. Proc Natl Acad Sci
USA (2002) 99:4495).
[0090] Expression of a nucleic acid sequence encoding a polypeptide
in the tissues of transgenic pigs can be assessed using techniques
that include, for example, Northern blot analysis of tissue samples
obtained from the animal, in situ hybridization analysis, Western
analysis, immunoassays such as enzyme-linked immunosorbent assays,
and reverse-transcriptase PCR (RT-PCR).
Vectors and Nucleic Acids
[0091] A variety of nucleic acids may be introduced into the
artiodactyl or other cells, for knockout purposes, or to obtain
expression of a gene for other purposes. Nucleic acid constructs
that can be used to produce transgenic animals include a target
nucleic acid sequence. As used herein, the term nucleic acid
includes DNA, RNA, and nucleic acid analogs, and nucleic acids that
are double-stranded or single-stranded (i.e., a sense or an
antisense single strand). Nucleic acid analogs can be modified at
the base moiety, sugar moiety, or phosphate backbone to improve,
for example, stability, hybridization, or solubility of the nucleic
acid. Modifications at the base moiety include deoxyuridine for
deoxythymidine, and 5-methyl-2'-deoxycytidine and
5-bromo-2'-doxycytidine for deoxycytidine. Modifications of the
sugar moiety include modification of the 2' hydroxyl of the ribose
sugar to form 2'-O-methyl or 2'-O-allyl sugars. The deoxyribose
phosphate backbone can be modified to produce morpholino nucleic
acids, in which each base moiety is linked to a six membered,
morpholino ring, or peptide nucleic acids, in which the
deoxyphosphate backbone is replaced by a pseudopeptide backbone and
the four bases are retained. See, Summerton and Weller (1997)
Antisense Nucleic Acid Drug Dev. 7(3):187; and Hyrup et al. (1996)
Bioorgan. Med. Chem. 4:5. In addition, the deoxyphosphate backbone
can be replaced with, for example, a phosphorothioate or
phosphorodithioate backbone, a phosphoroamidite, or an alkyl
phosphotriester backbone.
[0092] The target nucleic acid sequence can be operably linked to a
regulatory region such as a promoter. Regulatory regions can be
porcine regulatory regions or can be from other species. As used
herein, operably linked refers to positioning of a regulatory
region relative to a nucleic acid sequence in such a way as to
permit or facilitate transcription of the target nucleic acid.
[0093] Any type of promoter can be operably linked to a target
nucleic acid sequence. Examples of promoters include, without
limitation, tissue-specific promoters, constitutive promoters, and
promoters responsive or unresponsive to a particular stimulus.
Suitable tissue specific promoters can result in preferential
expression of a nucleic acid transcript in beta cells and include,
for example, the human insulin promoter. Other tissue specific
promoters can result in preferential expression in, for example,
hepatocytes or heart tissue and can include the albumin or
alpha-myosin heavy chain promoters, respectively. In other
embodiments, a promoter that facilitates the expression of a
nucleic acid molecule without significant tissue- or
temporal-specificity can be used (i.e., a constitutive promoter).
For example, a beta-actin promoter such as the chicken beta-actin
gene promoter, ubiquitin promoter, miniCAGs promoter,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, or
3-phosphoglycerate kinase (PGK) promoter can be used, as well as
viral promoters such as the herpes simplex virus thymidine kinase
(HSV-TK) promoter, the SV40 promoter, or a cytomegalovirus (CMV)
promoter. In some embodiments, a fusion of the chicken beta actin
gene promoter and the CMV enhancer is used as a promoter. See, for
example, Xu et al. (2001) Hum. Gene Ther. 12:563; and Kiwaki et al.
(1996) Hum. Gene Ther. 7:821.
[0094] An example of an inducible promoter is the tetracycline
(tet)-on promoter system, which can be used to regulate
transcription of the nucleic acid. In this system, a mutated Tet
repressor (TetR) is fused to the activation domain of herpes
simplex virus VP16 trans-activator protein to create a
tetracycline-controlled transcriptional activator (tTA), which is
regulated by tet or doxycycline (dox). In the absence of
antibiotic, transcription is minimal, while in the presence of tet
or dox, transcription is induced. Alternative inducible systems
include the ecdysone or rapamycin systems. Ecdysone is an insect
molting hormone whose production is controlled by a heterodimer of
the ecdysone receptor and the product of the ultraspiracle gene
(USP). Expression is induced by treatment with ecdysone or an
analog of ecdysone such as muristerone A. The agent that is
administered to the animal to trigger the inducible system is
referred to as an induction agent.
[0095] Additional regulatory regions that may be useful in nucleic
acid constructs, include, but are not limited to, polyadenylation
sequences, translation control sequences (e.g., an internal
ribosome entry segment, IRES), enhancers, inducible elements, or
introns. Such regulatory regions may not be necessary, although
they may increase expression by affecting transcription, stability
of the mRNA, translational efficiency, or the like. Such regulatory
regions can be included in a nucleic acid construct as desired to
obtain optimal expression of the nucleic acids in the cell(s).
Sufficient expression, however, can sometimes be obtained without
such additional elements.
[0096] A nucleic acid construct may be used that encodes signal
peptides or selectable markers. Signal peptides can be used such
that an encoded polypeptide is directed to a particular cellular
location (e.g., the cell surface). Non-limiting examples of
selectable markers include puromycin, ganciclovir, adenosine
deaminase (ADA), aminoglycoside phosphotransferase (neo, G418,
APH), dihydrofolate reductase (DHFR),
hygromycin-B-phosphtransferase, thymidine kinase (TK), and
xanthin-guanine phosphoribosyltransferase (XGPRT). Such markers are
useful for selecting stable transformants in culture. Other
selectable markers include fluorescent polypeptides, such as green
fluorescent protein or yellow fluorescent protein.
[0097] In some embodiments, a sequence encoding a selectable marker
can be flanked by recognition sequences for a recombinase such as,
e.g., Cre or Flp. For example, the selectable marker can be flanked
by loxP recognition sites (34-bp recognition sites recognized by
the Cre recombinase) or FRT recognition sites such that the
selectable marker can be excised from the construct. See, Orban, et
al., Proc. Natl. Acad. Sci. (1992) 89:6861, for a review of Cre/lox
technology, and Brand and Dymecki, Dev. Cell (2004) 6:7. A
transposon containing a Cre- or Flp-activatable transgene
interrupted by a selectable marker gene also can be used to obtain
transgenic animals with conditional expression of a transgene. For
example, a promoter driving expression of the marker/transgene can
be either ubiquitous or tissue-specific, which would result in the
ubiquitous or tissue-specific expression of the marker in F0
animals (e.g., pigs). Tissue specific activation of the transgene
can be accomplished, for example, by crossing a pig that
ubiquitously expresses a marker-interrupted transgene to a pig
expressing Cre or Flp in a tissue-specific manner, or by crossing a
pig that expresses a marker-interrupted transgene in a
tissue-specific manner to a pig that ubiquitously expresses Cre or
Flp recombinase. Controlled expression of the transgene or
controlled excision of the marker allows expression of the
transgene.
[0098] In some embodiments, the target nucleic acid encodes a
polypeptide. A nucleic acid sequence encoding a polypeptide can
include a tag sequence that encodes a "tag" designed to facilitate
subsequent manipulation of the encoded polypeptide (e.g., to
facilitate localization or detection). Tag sequences can be
inserted in the nucleic acid sequence encoding the polypeptide such
that the encoded tag is located at either the carboxyl or amino
terminus of the polypeptide. Non-limiting examples of encoded tags
include glutathione S-transferase (GST) and FLAG.TM. tag (Kodak,
New Haven, Conn.).
[0099] In other embodiments, the target nucleic acid sequence
induces RNA interference against a target nucleic acid such that
expression of the target nucleic acid is reduced. For example the
target nucleic acid sequence can induce RNA interference against a
nucleic acid encoding a cystic fibrosis transmembrane conductance
regulatory (CFTR) polypeptide. For example, double-stranded small
interfering RNA (siRNA) or small hairpin RNA (shRNA) homologous to
a CFTR DNA can be used to reduce expression of that DNA. Constructs
for siRNA can be produced as described, for example, in Fire et al.
(1998) Nature 391:806; Romano and Masino (1992) Mol. Microbiol.
6:3343; Cogoni et al. (1996) EMBO J. 15:3153; Cogoni and Masino
(1999) Nature 399:166; Misquitta and Paterson (1999) Proc. Natl.
Acad. Sci. USA 96:1451; and Kennerdell and Carthew (1998) Cell
95:1017. Constructs for shRNA can be produced as described by
McIntyre and Fanning (2006) BMC Biotechnology 6:1. In general,
shRNAs are transcribed as a single-stranded RNA molecule containing
complementary regions, which can anneal and form short
hairpins.
[0100] Nucleic acid constructs can be methylated using an SssI CpG
methylase (New England Biolabs, Ipswich, Mass.). In general, the
nucleic acid construct can be incubated with S-adenosylmethionine
and SssI CpG-methylase in buffer at 37.degree. C. Hypermethylation
can be confirmed by incubating the construct with one unit of
HinP1I endonuclease for 1 hour at 37.degree. C. and assaying by
agarose gel electrophoresis.
[0101] Nucleic acid constructs can be introduced into embryonic,
fetal, or adult artiodactyl cells of any type, including, for
example, germ cells such as an oocyte or an egg, a progenitor cell,
an adult or embryonic stem cell, a primordial germ cell, a kidney
cell such as a PK-15 cell, an islet cell, a beta cell, a liver
cell, or a fibroblast such as a dermal fibroblast, using a variety
of techniques. Non-limiting examples of techniques include the use
of transposon systems, recombinant viruses that can infect cells,
or liposomes or other non-viral methods such as electroporation,
microinjection, or calcium phosphate precipitation, that are
capable of delivering nucleic acids to cells.
[0102] In transposon systems, the transcriptional unit of a nucleic
acid construct, i.e., the regulatory region operably linked to a
target nucleic acid sequence, is flanked by an inverted repeat of a
transposon. Several transposon systems, including, for example,
Sleeping Beauty (see, U.S. Pat. No. 6,613,752 and U.S. Publication
No. 2005/0003542); Frog Prince (Miskey et al. (2003) Nucleic Acids
Res. 31:6873); Tol2 (Kawakami (2007) Genome Biology 8(Supp1.1):S7;
Minos (Pavlopoulos et al. (2007) Genome Biology 8(Supp1.1):S2);
Hsmar1 (Miskey et al. (2007)) Mol Cell Biol. 27:4589); and Passport
have been developed to introduce nucleic acids into cells,
including mice, human, and pig cells. The Sleeping Beauty
transposon is particularly useful. A transposase can be delivered
as a protein, encoded on the same nucleic acid construct as the
target nucleic acid, can be introduced on a separate nucleic acid
construct, or provided as an mRNA (e.g., an in vitro-transcribed
and capped mRNA).
[0103] Insulator elements also can be included in a nucleic acid
construct to maintain expression of the target nucleic acid and to
inhibit the unwanted transcription of host genes. See, for example,
U.S. Publication No. 2004/0203158. Typically, an insulator element
flanks each side of the transcriptional unit and is internal to the
inverted repeat of the transposon. Non-limiting examples of
insulator elements include the matrix attachment region-(MAR) type
insulator elements and border-type insulator elements. See, for
example, U.S. Pat. Nos. 6,395,549, 5,731,178, 6,100,448, and
5,610,053, and U.S. Publication No. 2004/0203158.
[0104] Nucleic acids can be incorporated into vectors. A vector is
a broad term that includes any specific DNA segment that is
designed to move from a carrier into a target DNA. A vector may be
referred to as an expression vector, or a vector system, which is a
set of components needed to bring about DNA insertion into a genome
or other targeted DNA sequence such as an episome, plasmid, or even
virus/phage DNA segment. Vector systems such as viral vectors
(e.g., retroviruses, adeno-associated virus and integrating phage
viruses), and non-viral vectors (e.g., transposons) used for gene
delivery in animals have two basic components: 1) a vector
comprised of DNA (or RNA that is reverse transcribed into a cDNA)
and 2) a transposase, recombinase, or other integrase enzyme that
recognizes both the vector and a DNA target sequence and inserts
the vector into the target DNA sequence. Vectors most often contain
one or more expression cassettes that comprise one or more
expression control sequences, wherein an expression control
sequence is a DNA sequence that controls and regulates the
transcription and/or translation of another DNA sequence or mRNA,
respectively.
[0105] Many different types of vectors are known. For example,
plasmids and viral vectors, e.g., retroviral vectors, are known.
Mammalian expression plasmids typically have an origin of
replication, a suitable promoter and optional enhancer, and also
any necessary ribosome binding sites, a polyadenylation site,
splice donor and acceptor sites, transcriptional termination
sequences, and 5' flanking non-transcribed sequences. Examples of
vectors include: plasmids (which may also be a carrier of another
type of vector), adenovirus, adeno-associated virus (AAV),
lentivirus (e.g., modified HIV-1, SIV or FIV), retrovirus (e.g.,
ASV, ALV or MoMLV), and transposons (e.g., Sleeping Beauty,
P-elements, Tol-2, Frog Prince, piggyBac).
[0106] As used herein, the term nucleic acid refers to both RNA and
DNA, including, for example, cDNA, genomic DNA, synthetic (e.g.,
chemically synthesized) DNA, as well as naturally occurring and
chemically modified nucleic acids, e.g., synthetic bases or
alternative backbones. A nucleic acid molecule can be
double-stranded or single-stranded (i.e., a sense or an antisense
single strand). The term transgenic is used broadly herein and
refers to a genetically modified organism or genetically engineered
organism whose genetic material has been altered using genetic
engineering techniques. A knockout artiodactyl is thus transgenic
regardless of whether or not exogenous genes or nucleic acids are
expressed in the animal or its progeny.
Example 1
Genetically Modified Artiodactyl Livestock (Bovine) Produced by
Direct Injection of TALENs
[0107] Three TALEN pairs were designed and assembled as described
in Cermak et. al. (2011) Nuc. Acids Res. 39:e82 (FIG. 3) and mRNA
was injected into the cytoplasm of bovine embryos at about 19 hours
post fertilization. Injected embryos were cultured in vitro and
collected at the blastocysts stage (FIG. 3). Individual blastocyst
genomic DNA was amplified by whole genome amplification (WGA)
(Carlson et al. (2011) Transgenic Res. 20:29) and screened for
indels using the SURVEYOR nuclease assay (FIGS. 3, 4A, and 4B).
Cleavage products indicative of TALEN activity were observed in 10%
of injected embryos (e.g., FIGS. 3, 4A, and 4B). Mutations in the
predicted region were confirmed in 2 blastocysts injected with
either ACAN11 or ACAN12 TALEN pairs (FIG. 3). A significant
decrease in the developmental competence of TALEN-injected embryos
was not observed. A second round of injections was then performed
using the ACAN12 TALEN pair at mRNA dosages ranging from 10-100
ng/.mu.l. Comparison of the blastocyst formation rate between
rounds 1 (33%) and 2 (5%) (10 ng/.mu.l conditions) revealed poor
embryo quality (Table S2). Despite the poor quality of the embryos,
12 putative mutants (27% of injected) were identified using the
SURVEYOR assay. The genotypes of each SURVEYOR positive embryo were
analyzed with 14 sequencing reads from cloned PCR products.
Sequencing revealed mosaicism in gene modification. Indels were
identified in 4 SURVEYOR positive embryos and of these, indel
positive sequence reads accounted for 7-29% of the total reads for
each embryo. These results demonstrated that TALENs functioned in
artiodactyl embryos. Gene modification of embryos with Zing Finger
Nucleases (ZFNs) and TALENs has been reported for model organisms
by direct injection of ZFN or TALEN mRNAs encoding a nuclease pair
Geurts et al. (2009) Science 325:433; Carbery et al., (2010)
Genetics 186:451; Mashimo et al. (2010) PLoS One 5:e8870; Tesson et
al. (2011) Nature Biotechnol. 29:695.
Example 2
Genetically Modified Artiodactyl Livestock (Swine) Produced by
Direct Injection of TALENs
[0108] A single set of injections was also conducted in porcine IVP
embryos using TALENs targeted to the porcine RELA gene (p65) for
which a tolerance allele for African Swine Fever has been proposed
(Palgrave et al. (2011) J. Virol. 85:6008). Two groups reported
enhancement in TALEN activity using truncated variants of the
native TALE scaffolds (Mussolino et al. (2011) Nucleic Acids Res.
39:9283; Miller et al. (2011) Nature Biotechnol. 29:143 {Mussolino,
2011 #151; Miller, 2011 #42}. Therefore, in contrast to experiments
above which used the +231 scaffold (BamHI fragment, Christian et.
al. (2010) Genetics 186:757), the p65 TALEN scaffold was chosen to
truncate to mimic the +63 version described in Miller et al. 2011
(op cit) (also see FIG. 5). Zygotes were injected with a mixture of
mRNA including 10 ng/.mu.l each for the left and right TALENs along
with 5 ng/.mu.l EGFP mRNA as an indicator of successful injection.
Seventy five EGFP positive zygotes were identified (of 214
injected, 35%) and subjected to WGA indel analysis. PCR
amplification was successful from 56 of the EGFP positives embryos,
and 16 of these (29%) revealed indels by SURVEYOR assay (FIG. 3) or
sequence analysis. Mosaicism appears to be reduced in the porcine
zygotes compared to cattle, in fact, one third of the mutants
(6/16) were either homozygous or heterozygous bi-allelic mutants
(FIG. 4). These results demonstrated that TALENs functioned in
artiodactyl embryos. Processes for the creation of animal founder
lines based on modified embryos are well known.
Example 3
Genetically Modified Artiodactyl Livestock Produced by Genetic
Modification of Bovine and Swine Somatic Cells
[0109] Several additional TALEN pairs were assembled for targets in
pigs and cattle chosen based on either biomedical or agricultural
relevance. Binding domains of six TALEN pairs were placed in the
context of two previously described TALEN scaffolds (+231,
Christian et. al. 2010 (op cit) and +63, Miller et. al. 2011 (op
cit)) (FIG. 5). Each TALEN pair was transfected into primary
livestock fibroblasts, and genome modification was measured at day
3 by the SURVEYOR assay (Guschin et al. (2010) Methods Mol. Biol.
649:247). The most active TALEN pairs, DMDE7.1 and ACAN12,
displayed cleavage of 38% and 25% of chromosomes, respectively, and
Sanger sequencing revealed an assortment of indels characteristic
of NHEJ mediated DNA repair (FIG. 5 and FIG. 6). The TALENs
scaffold had a significant effect on activity in fibroblasts. In
total, 4 of 6 loci targeted with the +63 isoform cleaved at 3.5% or
greater while only the DMDE7.1 TALEN pair cleaved above 1% in the
+231 scaffold (FIG. 5). As noted in previous studies Doyon et al.
(2010) Nature Methods 8:74; Miller, (2011) op. cit., a 72 hour
incubation at 30.degree. C. after transfection also had a positive
effect on activity, and was required for activity of 3 TALEN pairs.
The success rate for generating active TALEN pairs has been high.
Data collected up to the time of filing shows that 23 of 36 (64%)
TALEN pairs were detectably active (>1.0% NHEJ) at 15 genes
scattered across the pig and cow genome, on autosomes and both the
X and Y chromosomes (Table S3). Three quarters of the active pairs
cleaved with high efficiency (19-40%) with an average modification
level of 25%. Clonal processes for the creation of animal founder
lines based on modified fibroblasts are well known.
Example 4
Extended Culture and Indel Enrichment by Transposon
Co-Transfection
[0110] TALEN pairs were transfected into fibroblasts and cultured
cells for 14+ days with or without transposon co-selection prior to
measurement of modification levels. The results are summarized in
FIG. 7. At day zero (D0), cells are transfected with a mixture of
plasmids including an expression cassette for each TALEN (two
plasmids), a transposon encoding a selection marker (a third
plasmid, encoding puromycin, and a transposase-expression cassette
(fourth plasmid). The TALEN plasmids are the main component (4-fold
excess by mass) of each transfection. Transfected cells are
cultured for 3 days at either 30 or 37.degree. C. prior to
splitting, collection of a sample for SURVEYOR assay and re-plating
for extended culture +/- selection for transposon integration. All
cells are cultured at 37.degree. C. after day 3. Cells cultured for
14+ days are collected for SURVEYOR assay and cryopreserved for
downstream applications, i.e., SCNT. For comparison, other
fibroblasts were transfected by Nucleofection and percent NHEJ was
measured at day 3, and in day 14+ non-selected (NS) and selected
(S) populations. For comparison, fibroblasts were also transfected
using cationic-lipids.
Example 5
Isolation of Mono- and Bi-Allelic KO Clones
[0111] Transgenic primary fibroblasts can be effectively expanded
and isolated as colonies when plated with non-transgenic
fibroblasts (feeder-cells) at standard densities (>150
cells/cm.sup.2) and subjected to drug selection using the
transposon co-selection technique applied above (Carlson et al.
(2011) Transgenic Res. 20:1125). To evaluate this approach,
puromycin resistant colonies for cells treated with six TALEN pairs
were isolated and their genotypes evaluated by SURVEYOR assay or
direct sequencing of PCR products spanning the target site (FIGS.
8A and 8B). In general, the proportion of indel positive clones was
similar to predictions made based on day 3 modification levels.
Bi-alleic knockout clones were identified for 5 of 6 TALEN pairs,
occurring in up to 35 percent of indel positive cells (Table 1). In
the majority of examples (15 of 23), indels were homozygous (same
indel on each allele) rather than unique indels on each allele
suggesting that sister chromatid-templated repair is common (FIG.
9). Notably, among modified clones, the frequency of bi-alleic
modification (17-35%) for the majority of TALEN pairs exceed
predictions based on day 3 modification (10-15.6%) if chromosome
cleavages are treated as independent events. Previous studies with
ZFN (Kim et al. (2009) Genome Res. 19:1279; Lee et al. (2010)
Genome Res. 20:81; Perez et al. (2008) Nature Biotechnol. 26:808)
are in support of these results. These previous studies suggested
that bi-allelic modification occurs in nearly one-third of modified
cells independent of the nuclease activity level.
Example 6
Chromosomal Deletions and Inversions with TALENs
[0112] It was hypothesized that simultaneous delivery of two TALEN
pairs targeting the same chromosome could induce large chromosomal
deletions or inversions. The TALEN pairs, DMDE6 and DMDE7.1 were
tested because of their high activity and the fact that a high
percentage of Duchene's Muscular Dystrophy is caused by gross
deletions (Blake, 2002) such that a porcine model would match to
the human condition. The results are summarized in FIG. 10. Day 3
gene modification levels were high for each TALEN pair (24% for
DMDE6 and 23% DMDE7.1), albeit slightly lower that when either
TALEN pair was transfected individually (FIG. 10). To determine if
the sequence between the two TALEN pairs had been deleted, PCR was
attempted with primers spanning the TALEN target sites. If the 6.5
kb sequence had been removed, a band of .about.500 bp was expected,
whereas the wild type band of 7 kb would not be amplified with the
PCR conditions chosen. A band near 500 bp was observed in
replicates where both TALEN pairs were introduced, but was absent
when either TALEN pair was introduced alone (FIG. 10).
[0113] Next, the cell population was assayed for inversion events
by PCR across presumptive new 5' and 3' junctions. Products were
observed at the expected size for both the 5' and 3' junctions of
the presumptive inversion only when both TALEN pairs were
introduced (FIG. 10). To validate further that the inversions,
colonies were generated using the transposon co-selection strategy
and screened for deletion and inversion events. Both deletion and
inversion events were recovered with high frequency (10.3% and 4.1%
respectively; n>1000) (Table S4). Deletion and inversion events
occurred with remarkable fidelity. Forty one out of 43 of the
inversion positive colonies were positive at both the 5' and 3'
junctions. Finally, sequencing of PCR products confirmed both
deletion and inversion events with addition or deletion of very few
nucleotides at their junctions (FIG. 11, 12).
Example 7
TALEN-Induced Homologous Recombination Eliminates Need for Linked
Selection Markers
[0114] A mutant myostatin allele (an 11 bp deletion) from Belgian
Blue cattle was placed into the genome of wild-type Wagyu cattle
(Grobet et al. (1997) Nature Genet. 17:71) (FIG. 13). When
transfected alone, the btGDF8.1 TALEN pair cleaved up to 16% of
chromosomes at the target locus (FIG. 13). TALENs (btGDF83.1) and a
dsDNA template (BB-HDR) were designed to introduce an 11-bp
deletion in exon-3 of bovine GDF8 (Belgium Blue mutation) by DSB
induced homologous recombination. Half of the binding site for the
left TALEN was missing in the BB-HDR template, to make it resistant
to TALEN cleavage. A SURVEYOR assay demonstrated activity of
btGDF83.1 TALENs at both 37 and 30.degree. C. The PCR product used
for this assay was generated using primers b and b' (shown panel a)
of FIG. 13). The BB-HDR template was not included in these
replicates since it would confound estimates of btGDF83.1 activity.
Allele specific PCR demonstrated that HDR induction was dependent
on co-transfection of TALENs and the BB-HDR template. The PCR assay
was developed to specifically detect HDR modified GDF8 alleles
using primers c and c' (shown panel a) of FIG. 13). The 3' end of
primer c' spanned the 11-bp deletion, and cannot amplify the wild
type allele "wt". Five hundred cell equivalents were included in
each PCR reaction including the positive control "C". Percent HDR
was determined by comparative densitometry between experimental and
control reactions. Co-transfection with a supercoiled DNA template
harboring a 1623 bp DNA fragment from Belgian Blue cattle resulted
in a gene conversion frequency (HDR) of up to 5% as suggested by
semi-quantitative PCR at day 3, without selection for the desired
event (FIG. 13). These results demonstrated that TALENs can be used
to effectively place exogenous nucleic acid sequences in livestock,
including alleles--and without markers. To assess the frequency of
placement in individual colonies, the transposon co-selection
strategy was implemented to isolate and expand individual colonies
for DNA sequencing. Gene conversion using template from Belgian
Blue cattle was detected in 5 colonies out of 366 examined by PCR.
Amplification with primers outside the Belgian Blue HDR template
and sequencing confirmed the presence of the expected 11 bp
deletion in 4 of the colonies (FIG. 14).
Example 8
TALEN Mediated DNA Cleavage as a Target for HDR in Livestock
Cells
[0115] A TALEN pair (LDLR2.1) targeted to the fourth exon of the
swine low density lipoprotein receptor (LDLR) gene was
co-transfected with the supercoiled plasmid Ldlr-E4N-stop, which
contains homology arms corresponding to the swine LDLR gene and a
gene-trap enabling expression of Neomycin phosphotransferase upon
HDR (FIG. 15). After 3 days of culture PCR analysis revealed that,
even without antibiotic selection, a band corresponding to an HDR
event can be detected at the targeted locus (lane 4) at 30.degree.
C. Selection of populations of cultured cells for 14 days with
geneticin (G418) resulted in significant enrichment of HDR cells
(lanes 11 & 13). Cloning of such modified cells by somatic cell
nuclear transfer was performed, with surrogate sows presently
gestating the embryos.
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[0116] Patent applications, patents, publications, and journal
articles set forth herein are hereby incorporated herein by
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[0121] B. REISS, et al., RecA protein stimulates homologous
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Sequence CWU 1
1
128171DNABos taurus 1ccctcttcct ttcctccagg gatccctcct acatggcctc
ccactggaga agcaacagag 60gagcacacag a 71271DNAArtificial
SequenceIndel of sequence 1 2ccctcttcct ttcctccagg gatccctcct
acagggactc ccactggaga agcaacagag 60gagcacacag a 71366DNAArtificial
SequenceIndel of sequence 1 3ccctcttcct ttcctccagg gatccctcct
acctcccact ggagaagcaa cagaggagca 60cacaga 66471DNABos taurus
4ccctcttcct ttcctccagg gatccctcct acatggcctc ccactggaga agcaacagag
60gagcacacag a 71559DNAArtificial SequenceIndel of sequence 4
5ccttcttcct ttcctccagg gatccctccc actggagaag caacagagga gcacacaga
59666DNAArtificial SequenceIndel of sequence 4 6ccctcttcct
ttcctccagg gatccctcct acttcccact ggagaagcaa cagaggagca 60cacaga
66770DNASus 7gcccccccac acagctgagc ccatgctgat ggagtaccct gaggctataa
ctcgcttggt 60gacagggtcc 70891DNAArtificial SequenceIndel of
sequence 7 8gcccccccac acagctgagc cacaagtcga gggtctcaga gtcactgagt
actgagtgct 60gatggagtac cctgaggcta taactcgctt g 91966DNAArtificial
SequenceIndel of sequence 7 9gcccccccac acagctgagc cctgatggag
taccctgagg ctataactcg cttggtgaca 60gggtcc 661069DNAArtificial
SequenceIndel of sequence 7 10gcccccccac acagctgagc catgctgatg
gagtaccctg aggctataac tcgcttggtg 60acagggtcc 691165DNAArtificial
SequenceIndel of sequence 7 11gcccccccac acagctgagc ccaatggagt
accctgaggc tataactcgc ttggtgacag 60ggtcc 651265DNAArtificial
SequenceIndel of sequence 7 12gcccccccac acagctgagc acacaggagt
accctgaggc tataactcgc ttggtgacag 60ggtcc 651370DNASus 13gcccccccac
acagctgagc ccatgctgat ggagtaccct gaggctataa ctcgcttggt 60gacagggtcc
701469DNAArtificial SequenceIndel of sequence 13 14gcccccccac
acagctgacc catgctgatg gagtaccctg aggctataac tcgcttggtg 60acagggtcc
691571DNAArtificial SequenceIndel of sequence 13 15gcccccccac
acagctgagc ccattgctga tggagtaccc tgaggctata actcgcttgg 60tgacagggtc
c 711669DNAArtificial SequenceIndel of sequence 13 16gcccccccac
acagctgagc ccagctgatg gagtaccctg aggctataac tcgcttggtg 60acagggtcc
691767DNAArtificial SequenceIndel of sequence 13 17gcccccccac
acagctgagc ccatgatgga gtaccctgag gctataactc gcttggtgac 60agggtcc
671869DNAArtificial SequenceIndel of sequence 13 18gcccccccac
acagctgagc ccagctgatg gagtaccctg aggctataac tcgcttggtg 60acagggtcc
691965DNAArtificial SequenceIndel of sequence 13 19gcccccccac
acagctgagc ccaatggagt accctgaggc tataactcgc ttggtgacag 60ggtcc
652069DNAArtificial SequenceIndel of sequence 13 20gcccccccac
acagctgagc catgctgatg gagtaccctg aggctataac tcgcttggtg 60acagggtcc
692169DNAArtificial SequenceIndel of sequence 13 21gcccccccac
acagctgagc catgctgatg gagtaccctg aggctataac tcgcttggtg 60acagggtcc
692267DNAArtificial SequenceIndel of sequence 13 22gcccccccac
acagctgagc ccatgatgga gtaccctgag gctataactc gcttggtgac 60agggtcc
672369DNAArtificial SequenceIndel of sequence 13 23gcccccccac
acagctgagc ccatgtgatg gagtaccctg aggctataac tcgcttggtg 60acagggtcc
692470DNAArtificial SequenceIndel of sequence 13 24gcccccccac
acagctgagc catgcctgat ggagtaccct gaggctataa ctcgcttggt 60gacagggtcc
702566DNAArtificial SequenceIndel of sequence 13 25gcccccccac
acagctgagc cctgatggag taccctgagg ctataactcg cttggtgaca 60gggtcc
662697DNABos taurus 26tcgccttccc tcttcctttc ctccagggat ccctcctaca
tggcctccca ctggagaagc 60aacagaggag cacacagaag gcccttctgc aacggaa
972797DNAArtificial SequenceIndel of sequence 26 27tcgccttccc
tcttcctttc ctccagggat ccctcctaca tggcttggcc tcccactgga 60gaagcaacag
aggagcacac agaaggccct tctgcaa 9728106DNAArtificial SequenceIndel of
sequence 26 28tcgccttccc tcttcctttc ctccagggat ccctcctaca
tggctcccac tggctcccac 60tggagaagca acagaggagc acacagaagg cccttctgca
acggaa 1062996DNAArtificial SequenceIndel of sequence 26
29tcgccttccc tcttcctttc ctccagggat ccctcctaca tggctggcct cccactggag
60aagcaacaga ggagcacaca gaaggccctt ctgcaa 963092DNAArtificial
SequenceIndel of sequence 26 30tcgccttccc tcttcctttc ctccagggat
ccctcctacg cctcccactg gagagcaaca 60gaggagcaca cagaggccct tctgcaacgg
aa 923191DNAArtificial SequenceIndel of sequence 26 31tcgccttccc
tcttcctttc ctccagggat ccctcctact tcccactgga gaagcaacag 60aggagcacac
agaggccctt ctgcaacgga a 913289DNAArtificial SequenceIndel of
sequence 26 32tcgccttccc tcttcctttc ctccagggat ccctcctact
cccactggag aagcaacaga 60ggagcacaca gaggcccttc tgcacggaa
893391DNAArtificial SequenceIndel of sequence 26 33tcgccttccc
tcttcctttc ctccagggat ccctcctgct cccactggag aagcaacaga 60ggagcacaca
gaaggccctt ctgcaacgga a 913479DNAArtificial SequenceIndel of
sequence 26 34tcaccttcct tcttcctttc ctccagggat cccactggag
aagcaacaga ggagcacaca 60gaaggccctt ctgcacgga 793581DNAArtificial
SequenceIndel of sequence 26 35tcgccttccc tcttcctttc ctccaggcct
cccactggag aagcaacaga ggagcacaca 60gaaggccctt ctgcaacgga a
813671DNAArtificial SequenceIndel of sequence 26 36tcgccttccc
tcttcctttc ctccagggat ccctcacaga ggagcacaca gaaggccctt 60ctgcaacgga
a 713797DNAArtificial SequenceIndel of sequence 26 37tcgccttccc
tcttcctttc ctccagggat ccctcctaca ttgcctccca ctggagaagc 60aacagaggag
cacacagaag gcccttctgc aacggaa 973897DNAArtificial SequenceIndel of
sequence 26 38tcgccttccc tcttcctttc ctccagggat ccctcctaca
gggatcttcc cactggagaa 60gcacagagga gcacacagaa ggcccttctg caacgga
973917DNASus 39ctcctacaag tggattt 174053DNASus 40gacgggaaat
gcatctccta caagtgcacg aacagtccct ataaacccct gga 534153DNASus
41gacgggaaat gcatctccta caagtgcacg aacagtccct ataaacccct gga
534226DNASus 42gacgggaaat gcatctccta caagtg 264353DNAArtificial
SequenceIndel of sequence 40 43aaatgcatct cctacaagtg catttttgtt
taaattaaga tgtgggagga gaa 534454DNASus 44aaatgcatct cctacaagtg
catttttgtt taaattaaga tgtgggaagg agaa 544525DNAArtificial
SequenceIndel of sequence 40 45aaatgcatct cctacaagtg gattt
254629DNAArtificial SequenceIndel of sequence 40 46catctcctac
aagtgcaagg acgggtccg 294729DNAArtificial SequenceIndel of sequence
40 47catctcctac aagtgcaagg acgggtccg 294853DNAArtificial
SequenceIndel of sequence 40 48catctcctac aagtggattt gtgatgggaa
caccgagtgc aaggacgggt ccg 534923DNAArtificial SequenceIndel of
sequence 40 49ctcctacaag gacgggtccg atg 235023DNAArtificial
SequenceIndel of sequence 40 50ctcctacaag gacgggtccg atg
235153DNAArtificial sequenceIndel of sequence 40 51ctcctacaag
tggatttgtg atgggaacac cgagtgcaag gacgggtccg atg 535240DNAArtificial
SequenceIndel of sequence 40 52cctacaagtg gtttgtgagt gcaaggacgg
gtccgatgag 405340DNAArtificial SequenceIndel of sequence 40
53cctacaagtg gtttgtgagt gcaaggacgg gtccgatgag 405453DNAArtificial
SequenceIndel of sequence 40 54cctacaagtg gatttgtgat gggaacaccg
agtgcaagga cgggtccgat gag 535581DNASus 55wtgccacaca acgactggaa
catgcattca acatcgccaa gtatcagtta ggcatagaga 60aactactgga tcctgaaggt
t 815675DNAArtificial SequenceIndel of sequence 55 56gccacacaac
gactggaaca tgcattcaac atcgccatca gttaggcata gagaaactac 60tggatcctga
aggtt 755772DNAArtificial SequenceIndel of sequence 55 57gccacacaac
gactggaaca tgcattcaac atcgccagtt aggcatagag aaactactgg 60atcctgaagg
tt 725869DNAArtificial SequenceIndel of sequence 55 58gccacacaac
gactggaaca tgcattcaac atcagttagg catagagaaa ctactggatc 60ctgaaggtt
695972DNAArtificial SequenceIndel of sequence 55 59gccacacaac
gactggaaca tgcattcaac atcgccagtt aggcatagag aaactactgg 60atcctgaagg
tt 726079DNAArtificial SequenceIndel of sequence 55 60gccacacaac
gactggaaca tgcattcaac atcgccaagt atcagttagg catagagaaa 60ctactggatc
ctgaaggtt 796179DNASus 61gccacacaac gactggaaca tgcattcaac
atcgccaagt atcagttagg catagagaaa 60ctactggatc ctgaaggtt
796281DNAArtificial SequenceIndel of sequence 61 62cccacacaac
gactggaaca tgcattcaac atcgcataat atatcagtta ggcatagaga 60aactactgga
tcctgaaggt t 816377DNAArtificial SequenceIndel of sequence 61
63gccacacaac gactggaaca tgcattcaac atcgccaaat cagttaggca tagagaaact
60actggatcct gaaggtt 776469DNAArtificial SequenceIndel of sequence
61 64gccacacaac gactggaaca tgcattcaac atcagttagg catagagaaa
ctactggatc 60ctgaaggtt 696579DNAArtificial SequenceIndel of
sequence 61 65gccacacaac gactggaaca tgcattcaac atcgccaagt
atcagttagg catagagaaa 60ctactggatc ctgaaggtt 796676DNAArtificial
SequenceIndel of sequence 61 66gccacacaac gactggaaca tgcattcaac
atcgccaatc agttaggcat agagaaacta 60ctggatcctg aaggtt
766775DNAArtificial SequenceIndel of sequence 61 67gccacacaac
gactggaaca tgcattcaac atcgccaagt attaggcata gagaaactac 60tggatcctga
aggtt 756878DNASus 68gggaaatgca tctcctacaa gtggatttgt gatgggaaca
ccgagtgcaa ggacgggtcc 60gatgagtccc tggagacg 786982DNAArtificial
SequenceIndel of sequence 68 69gggaaatgca tctcctacaa gtggatttgt
gatgggggaa caccgagtgc agggacgggt 60cataagagtc cctggagacg tg
827074DNAArtificial SequenceIndel of sequence 68 70gggaaatgca
tctcctacaa gtggatttgt ggaacaccga gtgcaaggac gggtccgatg 60agtccctgga
gacg 747174DNAArtificial SequenceIndel of sequence 68 71gggaaatgca
tctcctacaa gtggatttgt ggaacaccga gtgcaaggac gggtccgatg 60agtccctgga
gacg 747274DNAArtificial SequenceIndel of sequence 68 72gggaaatgca
tctcctacaa gtggatttgt ggaacaccga gtgcaaggac gggtccgatg 60agtccctgga
gacg 747369DNAArtificial SequenceIndel of sequence 68 73gggaaatgca
tctcctacaa gtggatttgc accgagtgca aggacgggtc cgatgagtcc 60ctggagacg
697469DNAArtificial SequenceIndel of sequence 68 74gggaaatgca
tctcctacaa gtggacttgc accgagtgca aggacgggtc cgatgagtcc 60ctggagacg
697566DNAArtificial SequenceIndel of sequence 68 75gggaaatgca
tctcctacaa gtggatttgt gagtgcaagg acgggtccga tgagtccctg 60gagacg
667666DNAArtificial SequenceIndel of sequence 68 76gggaaatgca
tctcctacaa gtggatttgt gagtgcaagg acgggtccga tgagtccctg 60gagacg
667754DNAArtificial SequenceIndel of sequence 68 77gggaaatgca
tctcctacaa gtgcaaggac gggtccgatg agtccctgga gacg
547848DNAArtificial SequenceIndel of sequence 68 78gggaaatgca
tctcctacaa ggacgggtcc gatgagtccc tggagaag 487950DNAsus 79gggaaatgca
tctcctacaa gtggacgggt ccgatgagtc cctggagacg 508050DNAArtificial
SequenceIndel of sequence 79 80tgctgatcct ggcactgatc aaggacgggt
ccgatgagtc cctggagacg 508150DNAArtificial SequenceIndel of sequence
79 81gggaaatgca tctcctacaa gtggacgggt ccgatgagtc cctggagacg
508250DNAArtificial SequenceIndel of sequence 79 82tgctgatcct
ggcactgatc aaggacgggt ccgatgagtc cctggagacg 508341DNAArtificial
SequenceIndel of sequence 79 83tagacacagg gagtatggtc acttgctgat
tcccaccgag t 418469DNAArtificial SequenceIndel of sequence 79
84gggaaatgca tctcctacaa gtggatttgc accgagtgca aggacgggtc cgatgagtcc
60ctggagacg 698570DNAArtificial SequenceIndel of sequence 79
85gggaaatgca tctcctacaa gtggatttgt gatgggaaca ccgagtgcaa ggacgggtcc
60gatgagtccc 708677DNAArtificial SequenceIndel of sequence 79
86gggaaatgca tctcctacaa gtgcaaggag tgcaaggacg gaatgcaccg agtgcaagga
60cgggtccgat gagtccc 778756DNAArtificial SequenceIndel of sequence
79 87gggaaatgca tctcctacaa gttgcattcc gctgtgaatt aggatgtggg cggaga
568875DNASus 88gggaaatgca tctcctacaa gtggatttgt gatgacaccg
agtgcaagga cgggtccgat 60gagtccctgg agacg 758954DNAArtificial
SequenceIndel of sequence 89 89ggctcatttc tcagcttgca gtgcaaggac
gggtccggtg agtccctgga gacg 549076DNAArtificial SequenceIndel of
sequence 89 90gggaaatgca tctcctacaa gtggatttgt gatacacacc
gagtgcaagg acgggtccga 60tgagtccctg gagacg 769162DNASus 91tctataccta
ggtcaaaaat gtaatgaaga aagtatcagt taggcataga gaaactactg 60ga
629252DNAArtificial SequenceIndel of sequence 91 92tctataccta
ggtcaaaaat gtaattcagt taggcataga gaaactactg ga 529354DNAArtificial
SequenceIndel of sequence 91 93tctataccta ggtcaaaaat gtaatgatca
gttaggcata gagaaactac tgga 549462DNAArtificial SequenceIndel of
sequence 91 94tctataccta ggtcaaaaat gtaatgagac aggtatcagt
taggcataga gaaactactg 60ga 629544DNAArtificial SequenceIndel of
sequence 91 95tctataccta ggtcaaaaat gttaggcata gagaaactac tgga
449643DNAArtificial SequenceIndel of sequence 91 96tctataccta
ggtcaaaaat gtaggcatag agaaactact gga 439751DNAArtificial
SequenceIndel of sequence 91 97tctataccta ggtcaaaaat gtaatcagtt
aggcatagag aaactactgg a 519862DNAArtificial SequenceIndel of
sequence 91 98tctataccta ggtcaaaaat gtaatgaaga aagtatcagt
taggcataga gaaactactg 60ga 629961DNAArtificial SequenceIndel of
sequence 91 99tctataccta ggtcaaaaat gtaatgaaga aatatcagtt
aggcatagag aaactactgg 60a 6110062DNAArtificial SequenceIndel of
sequence 91 100tctataccta ggtcaaaaat gtaatgaatt aaatatcagt
taggcataga gaaactactg 60ga 6210160DNAArtificial SequenceIndel of
sequence 91 101tctataccta ggtcaaaaat gtaatgaaga gtatcagtta
ggcatagaga aactactgga 6010256DNAArtificial SequenceIndel of
sequence 91 102tctataccta ggtcaaaaat gtaaaggtat cagttaggca
tagagaaact actgga 5610363DNAArtificial SequenceIndel of sequence 91
103tctataccta ggtcaaaaat gtaaagaaaa ggggtatcag ttaggcatag
agaaactact 60gga 6310449DNAArtificial SequenceIndel of sequence 91
104tctataccta ggtcaaaaat tatcagttag gcatagagaa actactgga
4910554DNAArtificial SequenceIndel of sequence 91 105tctataccta
ggtcaaaaat gtaagaatca gttaggcata gagaaactac tgga 5410681DNASus
106gaaattttct ttctatacct atgtcaaaaa tgtaatgaaa cttggcgatg
ttgaatgcat 60gttccagtcg ttgtgtggct g 8110777DNAArtificial
SequenceIndel of sequence 106 107gaaattttct ttctatacct atgtcaaaaa
tgtaatgatg gcgatgttga atgcatgttc 60cagtcgttgt gtggctg
7710860DNAArtificial SequenceIndel of sequence 106
108gaaattttct
ttctatacct agggcgatgt tgaatgcatg ttccagtcgt tgtgtggctg
6010973DNAArtificial SequenceIndel of sequence 106 109gaaattttct
ttctatacct aggtcaaaaa tgtatggcga tgttgaatgc atgttccagt 60cgttgtgtgg
ctg 7311082DNAArtificial SequenceIndel of sequence 106
110gaaattttct ttctatacct aggtcaaaaa tgtaatgaag acttggcgat
gttgaatgca 60tgttccagtc gctgtgtggc tg 8211186DNAArtificial
SequenceIndel of sequence 106 111gaaattttct ttcgatacct aggtcaaaaa
tgtaatgaag atctacttgg cgatgttgaa 60tgcatgttcc agtcgttgtg tggctg
8611287DNAArtificial SequenceIndel of sequence 106 112gaaattttct
ttctatacct aggtcaaaaa tgtaatgaag atgatacttg gcgatgttga 60atgcatgttc
cagtcgttgt gtggctg 8711379DNAArtificial SequenceIndel of sequence
106 113ttttcactgt tagtttgttg caatccagcc atgatattca tcagttaggc
atagagaaac 60tactggatcc tgaaggtta 7911484DNAArtificial
SequenceIndel of sequence 106 114ttttcactgt tagtttgttg caatccagcc
atgatattct tagtatcagt taggcataga 60gaaactactg gatcctgaag gtta
8411579DNAArtificial SequenceIndel of sequence 106 115ttttcactgt
tggtttgttg caatccagcc aagatatata tcagttaggc atagagcaac 60tactggatcc
tgaaggtta 7911659DNAArtificial SequenceIndel of sequence 106
116tgtgatgaac actccacaga atctcgatgc tgtcgttacc ctctaactgt ggattttga
5911748DNAArtificial SequenceIndel of sequence 106 117tgtgacagaa
tctcgatgct gtcgttaccc tctaactgtg gattttga 4811859DNAArtificial
SequenceIndel of sequence 106 118ttgggcttga ttgtgacaga atctcgatgc
tgtcgttacc ctctaactgt ggattttga 5911911DNASus 119atgaacactc c
1112033DNASus 120cttgattgtg atgaacactc cacagaatct cga
3312122DNAArtificial SequenceIndel of sequence 120 121cttgattgtg
acagaatctc ga 2212222DNAArtificial SequenceIndel of sequence 120
122cttgattgtg acagaatctc ga 2212322DNAArtificial SequenceIndel of
sequence 120 123cttgattgtg acagaatctc ga 2212422DNAArtificial
SequenceIndel of sequence 120 124cttgattgtg acagaatctc ga
2212522DNAArtificial SequenceIndel of sequence 120 125cttgattgtg
acagaatctc ga 2212622DNAArtificial SequenceIndel of sequence 120
126cttgattgtg acagaatctc ga 2212722DNAArtificial SequenceIndel of
sequence 120 127cttgattgtg acagaatctc ga 2212822DNAArtificial
SequenceIndel of sequence 120 128cttgattgtg acagaatctc ga 22
* * * * *